2023 01 12 - 07 47 42 - FormA - VD3221x1e

Form A National no. Cantonal no.
Application for licence to perform animal experiment 32387 VD3221x1e

art. 18 Animal Welfare Act (SR 455), art. 141 Animal Welfare Ordinance (SR 455.1),
art. 30 Animal Experimentation Ordinance (SR 455.163)
Basics 01-08
01 Address of the applicant
Institute
Name EPFL SV IBI

Street EPFL SV CAV-GE SV 1839, Station 19
Postal code 1015
Town Lausanne
Company VD_EPFL
Resource manager
Name Julie Parchet-Piccand

E-mail julie.parchet-piccand@epfl.ch
Tel. no +41 21 693 73 42
Study Director
Name Kristina Schoonjans

E-mail kristina.schoonjans@epfl.ch
Tel. no +41216931891
02 Address of the cantonal authority
Name VD
Street Chemin du Marquisat 1
Postal code 1025
Town Saint-Sulpice
Delegated application input by the No
canton in animex-ch
03 Intercantonal experiment
Will the experiment be performed in No

more than one canton?
If yes: Secondary canton(s) none
Exported from animex-ch at: 12.01.2023 07:47:41 1 / 57

Application for animal experiment 32387 VD3221x1e
04 Title of application
Indication of application title Study of the role of Slc25a47 in hepatic metabolism
05 Title for the publication
Informative title to be used for the publication according to art. 20 let. a Animal Welfare Act (SR 455) after the end of the experiment.
Application title used for Study of the role of Slc25a47 in hepatic metabolism
thepublication
06 Application type
Corresponding application type Supplementary application
07 Maximum prospective degree of severity
Indication of the maximum 2

prospective degree of severity of this
application
Questions/ Answers
Submitted to institute - 14.04.2020 18:06:28
Pourriez-vous vérifier que le fiches AGM sont bien celles qui correspondent aux lignées que vous comptez utiliser ici?
Answered - 24.04.2020 09:45:11
Please find attached the updated GMA forms.
08 Duration of project and date of start
Duration of project 3 years / 0 months / 0 days

Date of proposed start 25.07.2020

Animals 9-10
09 Animal list
Animal List
Category info
Animal category: Mice gm
Sex: Mixed
Use of genetically modified animals: Yes
Has this line been newly established in this facility especially for this experiment?: No
Constrained Line: No
Data sheet info and related forms
Documents
 Name: GMA forms 3221.1 combined.pdf
 Description: Datasheets initially submitted in answers but mandatory here for the SC
Number info
Previously approved: 1834
Requested number of animals: 72
Total number of animals requested: 1906
Origin of the animals

 Origin type: Approved animal facility (incl. own breeding)
 Origin ID: From Switzerland / National number / name of animal facility: 127-VD-H11 EPFL CPG Rodents /
Address of animal facility: Station 19
Place where the animals are kept

 Place type: Approved animal facility (incl. own breeding)
 Place ID: National number / name of animal facility: 127-VD-H11 EPFL CPG Rodents / Address of animal
facility: Station 19 / Room numbers:
Category info
Animal category: Mice
Sex: Male
Use of genetically modified animals: No
Has this line been newly established in this facility especially for this experiment?: No
Constrained Line: No
Data sheet info and related forms
Documents
 No Records
Number info
Previously approved: 0
Requested number of animals: 60
Total number of animals requested: 60
Origin of the animals

 Origin type: Approved animal facility (incl. own breeding)
 Origin ID: From Switzerland / National number / name of animal facility: 127-VD-H11 EPFL CPG Rodents /
Address of animal facility: Station 19
Place where the animals are kept

 Place type: Approved animal facility (incl. own breeding)
 Place ID: National number / name of animal facility: 127-VD-H11 EPFL CPG Rodents / Address of animal
facility: Station 19 / Room numbers:

10 Location of the experiments
Address Room number Description

EPFL, SV/UDP, station 19, 1015
Lausanne

Personnel 11-14
11 Personnel details
Name Role Qualification Deputy Area of responsibility

status Study
Director
Julie Parchet-Piccand Resource Manager Resource manager
(RM)
Kristina Schoonjans Study Director of responsable de l'experience
Institute (SDI)
Johan Auwerx Study Director of directeur de l'experience suppleant
Institute (SDI)
Penelope Stefanelli Involved Person of procedures
Institute (IPI)
Francesca Pontanari Involved Person of procedures
Institute (IPI)
Alessia Perino Involved Person of procedures
Institute (IPI)
Sabrina Bichet Involved Person of procedures
Institute (IPI)
Antoine Jalil Involved Person of procedures
Institute (IPI)
Carla Ferreira Involved Person of procedures
Institute (IPI)
Hadrien Demagny Involved Person of procedures
Institute (IPI)
Jéromine Imbach Involved Person of procedures
Institute (IPI)
Thibaud Clerc Involved Person of procedures
Institute (IPI)
Fabiana Pereira Da Involved Person of procedures
Costa Fraga Institute (IPI)
Alejandro Alonso Involved Person of procedures
Calleja Institute (IPI)
Adrien Faure Involved Person of Procedures
Institute (IPI)
12 Resource Manager

Statement of responsibility The Resource Manager confirms that the persons named in the list
of persons are familiar with the regulations of the Animal Welfare
Act (SR 455) and Animal Welfare Ordinance (SR 455.1) applicable
to animal experiments and that they satisfy the educational and
further training requirements.
13 Principal Study Director
Name Kristina Schoonjans

Statement of responsibility The Principal Study Director confirms his / her responsibility as
stated in art. 131 Animal Welfare Ordinance (SR 455.1).

Name of Deputy Study Director Johan Auwerx
14 Animal Welfare Officer of Institute

Statement of responsibility By submitting to the cantonal veterinary office, the Animal Welfare
Officer confirms that the application has been completed in full and
contains the information required to assess its necessity (art. 129
let. a Animal Welfare Ordinance (SR 455.1)).
Timestamp of the submission 09.01.2023 09:12:11

Purpose of the experiment 15-22

In Sections 16-18, mark only one entry in each category and, where appropriate, enter a further mark in a sub-category.
15 Field of study
Indication of the scientific field Physiology
16 Area of application
The project is associated with Biological (including medical) studies in the field of basic research
following area of application
17 Association with diseases or disorders
The project is associated with Human diseases:

following diseases or disorders Cancer (excluding carcinogenicity tests)
18 Associated procedures required by law
Indication of the regulatory No association with procedures required by law

requirements
19 External expertise
Has the project been appraised? Yes

If yes: Expertise rendered by This project is partially funded by the Swiss Cancer League (KFS-
3444-08-2014) and by the European Commission (H2020-MSCA-
IF-2018 ; Grant no 846001) Other grants are being submitted to
finance this project.
20 Objective of the experiment and background
Brief description of the objective of the experiment (maximum one page). To assess compliance with the indispensability (see
section 38 to 40) of the experiment according to art. 17 Animal Welfare Act (SR 455): (1) Description of the aim, (2) Current state of
research, (3) Anticipated knowledge to be gained.
In the license No.VD3221, we explored the role of LRH-1 and its downstream targets, ASNS and Slc25a47,
using specific knockout mice strains. During the past 3 years, we have produced some important and interesting
research results. However, we have not finished the whole project. Therefore, we wish to continue our project in
the upcoming years, which still requires some of the previous listed experiments and mice. In parallel, we will
remove some mouse strains from this license. Indeed, preliminary data concerning the Asns study (ASNS-/-
mice) indicated no relevant phenotype for our project. Additionally, we will not make use of the LHR1hep-/- and
LHR1K289R mice in our further investigations, so now we plan to focus our research only on the more promising
SLC25A47 protein using the specific knockout mouse line.

Background:
One of the main functions of the liver is to coordinate and fine-tune the energy demands of
peripheral tissues. Disorders affecting hepatic metabolism, and more specifically, liver
mitochondria directly influence fatty acid oxidation and oxidative phosphorylation (OXPHOS),
resulting in impaired bile flow, liver steatosis, fibrogenesis and cell death. The metabolic flexibility
of hepatocytes is surprisingly high allowing the liver to cope with major disturbances, however,
this can be disrupted upon continuous metabolic stress. For instance, in the context of obesity and
insulin resistance, significant alterations occur favoring hepatic triglyceride accumulation
(steatosis) causing decreased mitochondrial bioenergetics and possibly leading to pathological
states such as nonalcoholic fatty liver disease (NAFLD). The solute carrier 25 family, SLC25, in
mammals is a superfamily of 53 nuclear-encoded proteins, mostly localized in the inner
mitochondrial membrane (IMM), that transport a variety of anions, nucleotides and metabolites
bridging biochemical processes occurring outside and inside the mitochondria. Due to the plethora
of molecules transported into and out of the mitochondria, SLC25 are involved and dictate many
metabolic pathways, such as oxidative phosphorylation, citric acid cycle, fatty acid oxidation,
amino acid degradation, gluconeogenesis and urea cycle. Slc25a47 is a member of the SLC25
family that was first cloned and identified in 2004 by sequencing specific clones downregulated in
a patient with hepatocellular carcinoma (HCC), thus being also named hepatocellular carcinoma
downregulated mitochondrial carrier protein (HDMCP). This observation highlights the expression
and function of this carrier as a hallmark of liver identity. However, SLC25A47 has been
completely understudied and currently nothing is known about its structure, function or solute
specificity, making it an interesting target of research. Considering that Slc25a47 expression
decreases in liver diseases, such as during HCC, the full characterization of this transporter could
aid to better understand its role in liver function and achieve better therapeutic strategies for liver
metabolic diseases.
Previous experiments conducted under the license No. VD3221 unraveled the crucial role of
SLC25A47 in liver physiology and general metabolic homeostasis. Indeed, preliminary data
obtained from the non-invasive metabolic phenotyping of the Slc25a47hep-/- mice (aim A2 and A3
of the parental license) revealed that SLC25A47 is helping in maintaining the metabolic
homeostasis of the liver and its loss may induce organismal compensatory responses. In order to
further investigate these phenomena and clarify the function of this important liver transporter, we
aim to perform functional assays and omics analysis in Slc25a47hep-/- and Slc25a47hep+/+ mice.
In addition, and since the Slc25a47hep-/- mice present confounding compensatory factors (like
the upregulation of fibroblast growth factor 21, FGF21), we will study the postulated intermediate
phenotype of the SLC25A47 heterozygous mice (Slc25a47hep+/-) and the loss of hepatic FGF21
in Slc25a47hep-/- mice, by using a newly generated mouse line (Fgf21hep-/- ; Slc25a47hep-/-
double KO mice).
References:
W. Lee and R. Sokol, "Liver Disease in Mitochondrial Disorders," Semin. Liver Dis., vol. 27, no. 3,
pp. 259-273, Aug. 2007.

N. E. Sunny, F. Bril, and K. Cusi, "Mitochondrial Adaptation in Nonalcoholic Fatty Liver Disease:
Novel Mechanisms and Treatment Strategies," Trends Endocrinol. Metab., vol. 28, no. 4, pp. 250-
260, 2017.
C. Koliaki et al., "Adaptation of Hepatic Mitochondrial Function in Humans with Non-Alcoholic

Fatty Liver Is Lost in Steatohepatitis," Cell Metab., vol. 21, no. 5, pp. 739-746, 2015.
F. Palmieri, "The mitochondrial transporter family (SLC25): physiological and pathological

implications," Pflgers Arch. Eur. J. Physiol., vol. 447, no. 5, pp. 689-709, Feb. 2004.
F. Palmieri and M. Monne¿, "Discoveries, metabolic roles and diseases of mitochondrial carriers:
A review," Biochim. Biophys. Acta - Mol. Cell Res., vol. 1863, no. 10, pp. 2362-2378, Oct. 2016.
M. G. K. Tan, L. L. P. J. Ooi, S. E. Aw, and K. M. Hui, "Cloning and identification of hepatocellular

carcinoma down-regulated mitochondrial carrier protein, a novel liver-specific uncoupling protein,"
J. Biol. Chem., vol. 279, no. 43, pp. 45235-45244, 2004.
Amendment January 2023
We recently published a paper (Bresciani et al. 2022). In this paper, we described the phenotype
of a new line (Slc25a47hep-/-). During the revision of the manuscript, we were asked whether
recombination of the loxP sites could affect the expression not only of Slc25a47, but also of Wars,
an important gene located downstream of Slc25a47. Concerns were raised on whether the
phenotypes observed in Slc25a47hep-/- mice were due to the downregulation of Wars rather than
Slc25a47. To clarify this, we would like to use two approaches. First, we would like to induce the
recombination by means of AAV8_hAAT-Cre in Slc25a47lox/lox mice and attempt to rescue the
phenotype by overexpression of either Slc25a47 or Wars. Second, we would like to silence
Slc25a47 in C57BL/6J mice using AAV8-short hairpin RNA. The first experiment would allow us to
understand which phenotypes are driven by Slc25a47. Whereas the second experiment would
give us the possibility of understanding the effects of Slc25a47 silencing in a model that is not
based on genomic recombination.
Some preliminary data shows that once SLC25A47 has matured, it is subjected to post-
translational cleavage. However, this modification is not observed in cellular models. Therefore,
we hypothesize that the physiological environment of the liver is necessary for these
modifications. Hence, we would like to express Slc25a47-Flag tagged in C57BL/6J mice by
means of AAV8_hAAT delivery specifically in hepatocytes. This approach would allow us to
immunoprecipitate the native form of SLC25A47 for further structural and functional studies.
Bresciani, N., Demagny, H., Lemos, V., Pontanari, F., Li, X., Sun, Y., ... & Schoonjans, K. (2022). The Slc25a47
locus is a novel determinant of hepatic mitochondrial function implicated in liver fibrosis. Journal of Hepatology,
77(4), 1071-1082.
Document(s): 0

21 Results of the previous application
Maximum one page must be completed in the case of renewal applications. Brief summary of the results of the previous licensing
procedure including the number of animals used, the degree of severity, and the rationale for renewal of the application.
Document(s): 0
22 Hypotheses
Formulate the research question to be answered in the experiment or the hypothesis (hypotheses) to be tested(confirmatory study).
The goals of this study will be: 1) To study the role of Slc25a47 in liver physiology and
metabolism. 2) To assess the role of Slc25a47 in the systemic metabolic homeostasis. 3) To
unravel the molecular pathways upstream and downstream of Slc25a47 in the hepatocytes.
We will make use of one mouse model that had been used in our lab for the past years and one
that was recently developed:
(1) The liver-specific SLC25A47 knockout mouse (Slc25a47hep-/-; with Slc25a47hep+/+ and
heterozygous Slc25a47hep+/-, as control);
(2) The liver-specific double knockout mouse, lacking both SLC25A47 and FGF21 (Fgf21hep-/-
;Slc25a47hep-/ DKO-).
SC-32387-VD3221x1d - Last submitted to canton: 30.06.2022
Amendment May 2022
In this amendment, we would like to introduce four new mouse models: 1) To validate the liver
specific and systemic phenotype. 2) To unravel the solute transported by SLC25A47. 3) To
understand the specific role of SLC25A47 in mitochondria.
We will make use of two mouse models which have already been used in our lab for the past
years and four new recently developed ones:
(1) The liver-specific SLC25A47 knockout mouse (Slc25a47hep-/-

; with Slc25a47hep+/+ and heterozygous Slc25a47hep+/-, as control – line name:
Slc25a47_tm1c_AlbCre);
(2) The liver-specific double knockout mouse, lacking both SLC25A47 and FGF21 (Fgf21hep-/-
; Slc25a47hep-/- DKO – line name: Fgf21_lox_ Slc25a47_lox_AlbCre);
(3) The whole body knock out mouse, lacking SLC25A47 in any cell type (Slc25a47CMV-/- - line
name: Slc25a47_tm1c_CMVCre);

(4) The inducible hepatocyte specific knock out mouse, lacking SLC25A47 after tamoxifen
induction specifically in hepatocytes (indSlc25a47hep-/- - line name:
Slc25a47_tm1c_Alb_IRES_CreERT2);
(5) The inducible whole body knock out mouse, lacking SLC25A47 after tamoxifen induction in
any cell type (indSlc25a47CMV-/- - line name: Slc25a47_tm1c_CAGG_CreERTM);
(6) The MITO-Tag hepatocyte specific knock out mouse, which present and HA-tag on their
hepatocytes’ mitochondria (Slc25a47MITOhep-/- - line name: Slc25a47_tm1c_AlbCre_MitoTag)
In this amendment, we would like to 1) acutely delete Slc25a47 in Slc25a47lox/lox mice by means of
silencing using AAV8_hAAT-Cre and co-expressing Slc25a47 or Wars to validate which gene is
the driver of the phenotype. 2) confirm that the glucose phenotype observed in Slc25a47hep-/- mice
is driven by Slc25a47. 3) validate the line by using short-hairpin RNAs against Slc25a47 to study
which phenotypes can be recapitulated when Slc25a47 is downregulated. 4) study SLC25A47
protein and its possible in vivo maturation, by means of FLAG immunoprecipitation. To this end,
we would like to use AAV8 vectors to overexpress Slc25a47-FLAG tagged in vivo.
(1) (2) The Slc25a47lox/lox mice (Slc25a47hep+/+, Slc25a47lox/lox; AlbCre 0 mice), present loxP sites
around Slc25a47 exon 5-6. Once mice are injected with AAV8_hAAT-Cre, the viral vector will
induce hepatocyte specific DNA recombination (because the viral vector is targeted to the liver)
and delete Slc25a47. Since these mice are non-carrier for the AlbCre transgene, AAV8_hAAT-Cre
will be used to induce acutely the deletion.
(3) (4) C57BL6/J wildtype mice, these mice will be used to express an shRNA construct to acutely
downregulate Slc25a47 and study its downregulation specific phenotypes. Moreover, these mice
will also be used to allow the immunoprecipitation of SLC25A47, by using AAV8_hAAT-Slc25a47
FLAG constructs.
Document(s): 0
Questions/ Answers
Since liver specific knock-out are smaller and their food intake is higher, do you expect a more severe phenotype in the
whole-body KO despite the fact that Slc25a47 is mainly expressed in the liver? Have you already observed homozygote
animals? How many of the 88 animals observed were homozygotes?

Questions/ Answers
Answered - 30.06.2022 12:06:02
Thank you for your question. Slc25a47 is mainly expressed in the liver, and the knock-out model we have been
using so far achieves 90-95% deletion of Slc25a47, these mice are in good health, they reproduce well, can
survive to over 80 weeks of age and do not present any particular sign of distress. Hence, we do not expect the
general well-being of the CMV mice to differ from the AlbCre model. However, we expect the liver phenotype to be
more pronounced and we hope to avoid blurring effects due to remaining expression of Slc25a47.
Concerning the mice generated so far, we have obtained 17 homozygote mice. 13 were euthanised between 12-
18 weeks of age for a revision (VD3290.1), 1 is 33 weeks old and 3 are below 10 weeks. The mice are in the
breeding facility and no anomaly has been picked up by the caretakers. During the revision of our manuscript
where we investigated the liver-specific role of Slc25a47, accepted in Journal of hepatology (doi:
10.1016/j.jhep.2022.05.040), a question arose of whether neighbouring genes were flanked by AlbCre
recombinase, and whether this could account for off-target effects. To validate the absence of off-target effects,
we used Slc25a47_tm1c_CMVCre mice and analyse neighbouring genes expression in multiple tissues
(epidydimal white adipose tissue, brain, quadriceps, kidney and liver). As hoped, there was no effect. During this
study, we observed that the Slc25a47_tm1c_CMVCre mice respect Mendelian ratio and show no sign of distress.
As expected Slc25a47_tm1c_CMVCre were smaller and Slc25a47 was indeed absent in the liver. We did not
investigate the line further since the mice were not aged matched, had different sexes and the liver was snap
frozen. Therefore, we cannot already conclude whether the liver phenotype has worsened with a constitutive
deletion of Slc25a47. However, we can say that the general wellbeing of the animals is not affected by Slc25a47
constitutive deletion.

Course of the experiment (Method I) 23-27

23 Course of the experiments: Schematic representation
Description of the course of the experiment from a temporal perspective. Representation of the course of the experiment or individual
steps (e.g. flow chart, workflow diagram, table). To include: animal model, animal groups, the overall duration of the experiment for each
group.
Schematic representation:
Except for the isolation of primary hepatocytes, we will use only male mice because estrogen regulates the
expression of Shp (Small heterodimer partner or Nr0b2), a corepressor of LRH-1, which is a direct regulator of
SLC25A47.
We designed two studies to understand the function of Slc25a47 in hepatic metabolism under
normal physiological conditions as well as pathological conditions. Study A: Assess the function of
Slc25a47 in hepatic metabolism. Study B: Elucidate the molecular pathway upstream and
downstream of Slc25a47 in hepatocytes.
Study A: Assess the function of Slc25a47 in hepatic metabolism The goal of this study will be
to define the function of Slc25a47 in liver metabolism. As mentioned above, our preliminary
findings unraveled the crucial role of SLC25A47 in liver homeostasis. Considering our data, we
hypothesize that the mitochondrial carrier could be involved in controlling lipid metabolism (e.g.,
beta oxidation). To assess this, we will use different mouse models to achieve this goal.
Study A1: Study the role of Slc25a47 in liver physiology and pathology (1) (Figure 1) In order to
further investigate the role played by SLC25A47 in liver metabolism, we will perform both omics
analyses (RNA sequencing, proteomics, metabolomics) and functional assays (such as oxygen
consumption assessments) in livers of Slc25a47hep-/- and control mice (Slc25a47hep+/+ and
Slc25a47hep-/+). Mice will be euthanized under overnight fasting (14h) or fed (CHOW diet)
condition by brief exposure to CO2 followed by cardiac puncture for blood collection and organs
harvesting.
Study A2: Study the role of Slc25a47 in liver physiology and pathology (2) (Figure 2)
We would like to complete the study A3 of the original license VD3221. Indeed, we have already
performed a non-invasive metabolic phenotyping of the mice (Slc25a47hep-/- and
Slc25a47hep+/+) on normal chow diet (CD); however, to better dissect the role of Slc25a47 in
hepatic metabolism in a pathological condition, we aim to take advantage of a model of liver
damage triggered by high-fat high-sucrose (HFHS) diet. Slc25a47hep-/- ; Slc25a47hep+/- and
Slc25a47hep+/+ mice will be then tested for: body composition (EchoMRI), energy expenditure
(by indirect calorimetry with Comprehensive Lab Monitoring System - CLAMS), food intake, feces
composition, glucose tolerance (by glucose tolerance test - GTT), insulin resistance (by insulin
tolerance test - ITT). Additionally, the mice will undergo a pyruvate tolerance test and a non-lethal
cold test.
Study A3: Study the solute carried by Slc25a47 (Figure 3) As previously mentioned, preliminary
studies conducted under the parental license VD3221 highlighted an interesting phenotype for the
Slc25a47hep-/- mice. In order to fully characterize this peculiar transporter and investigate the
carried metabolite(s), we plan to acutely downregulate Slc25a47 by transducing Slc25a47hep+/+

mice with AAV8-Cre before executing metabolomics analysis. This approach will allow the
investigation of the very first events and metabolic changes occurring in the liver upon
downregulation of the carrier. Moreover, it will narrow down the pathways most affected by this
loss and provide us with some candidates to test in vitro (transport assays). We will inject 8-12
weeks Slc25a47hep+/+ mice with AAV8-Cre or AAV8-Empty via tail vein. 5 weeks after injection
mice will be euthanized by brief exposure to CO2 followed by cardiac puncture for blood collection
and organs harvesting.
Study A4: Investigate the repercussions of the loss of Slc25a47 (Figure 4) Preliminary data
obtained under the license No VD3221 revealed that SLC25A47 is playing a crucial role in
maintaining the metabolic homeostasis of the liver and its expression, in normal conditions, is so
high that it can be considered as a hallmark of liver identity. Surprisingly, our observations showed
that 8 weeks old Slc25a47hep-/- mice express some patches of SLC25A47, suggesting possible
events of liver regeneration. In order to elucidate this phenomenon, we will compare the livers of
Slc25a47hep-/- and Slc25a47hep+/+ mice at different ages (day 0 and 4, 8, 33, 80 weeks old).
Mice will be euthanized after overnight fasting (14h) by brief exposure to CO2 followed by cardiac
puncture for blood collection; organs will be harvested.
Study A5: Rescue of the Slc25a47hep-/- mice phenotype by mean of metabolite supplementation
in drinking water (Figure 5). Our data showed that Slc25a47hep-/- mice present impaired
mitochondrial functions and accumulate triglycerides in the liver, a condition that resembles
NAFLD. Betaine is a naturally occurring dietary compound that is also synthesized in vivo from
choline. In vivo, betaine acts as a methyl donor for the conversion of homocysteine to methionine
and it also functions as an osmolyte. Oral betaine treatment has been evaluated in the treatment
of alcoholic liver disease; in addition, in animal models of NAFLD, betaine treatment improved
insulin and glucose levels and decreased indexes of steatosis. Similarly, choline (the direct
precursor of betaine) can rescue a hepatic steatosis phenotype. Taurine is a sulfur-including
amino acid that is present in mammalian tissues in minute levels. It is endogenously synthesized
from methionine and cysteine metabolism and is also provided in diets comprising fish and
shellfish. Taurine is involved in various physiological functions, including bile salt conjugation,
calcium homeostasis, osmoregulation, membrane stabilization, added to its anti-oxidant and
immunomodulatory effects. Interestingly, taurine administration can inhibit NAFLD in rodents by
reducing lipotoxicity, mitigating the oxidative stress and decreasing inflammation. We, therefore,
plan to ameliorate the steatotic phenotype of the Slc25a47hep-/- mice by treating them with
betaine, choline or taurine. 3 weeks old Slc25a47hep-/- and Slc25a47hep+/+ mice will receive 2%
betaine, 2% choline or 5% taurine (in the drinking water) or normal water for 5-6 weeks. The water
intake and the mice body weight will be periodically checked during the period of treatment. Mice
will be euthanized by brief exposure to CO2 followed by cardiac puncture for blood collection;
organs will be harvested.
References:
Murakami S., "Role of taurine in the pathogenesis of obesity", Mol Nutr Food Res 2015; 59:1353-
1363
Elango Kathirvel, Kengathevy Morgan, Ganesh Nandgiri,Brian C. Sandoval, Marie A. Caudill,

Teodoro Bottiglieri,Samuel W. French and Timothy R. Morgan,"Betaine improves nonalcoholic
fatty liver and associated hepatic insulin resistance: a potential mechanism for hepatoprotection
by betaine", Am J Physiol Gastrointest Liver Physiol, 299(5): G1068-G1077, 2010.

Rats Azza H. Abd Elwahaba, Amina M. Tolbac, Basma K. Ramadana, Mona F. Schaalanb," A
Novel Role of SIRT1/ FGF-21 in Taurine Protection Against Cafeteria Diet-Induced Steatohepatitis
in Rats", Cell Physiol Biochem, 43:644-659, 2017.
Al Rajabi A1, Castro GS, da Silva RP, Nelson RC, Thiesen A, Vannucchi H, Vine DF, Proctor SD,
Field CJ, Curtis JM, Jacobs RL., "Choline supplementation protects against liver damage by
normalizing cholesterol metabolism in Pemt/Ldlr knockout mice fed a high-fat diet.", J Nutr. 2014
Mar;144(3):252-7.
Study A6: Rescue of the Slc25a47hep-/- mice phenotype by mean of an Adeno-Associated viral
particles (AAV8)-mediated gene delivery (Figure 6) Moreover, we aim to rescue the Slc25a47hep-
/- mice phenotype by Adeno-Associated viral particles (AAV8)-mediated gene delivery. We will
inject 8-12 weeks Slc25a47hep+/+ and Slc25a47hep-/- mice with AAV8-Slc25a47 or AAV8-Empty
via tail vein. 5 weeks after injection mice will be euthanized by brief exposure to CO2 followed by
cardiac puncture for blood collection and organs harvesting.
Study A7: The role of SLC25A47 in the systemic metabolic homeostasis (Figure 7) Among the
known hepatokines, FGF21 is an important regulator of metabolism acting both as an
autocrine/paracrine factor and as a hormone released in the bloodstream. FGF21 is mainly
expressed in the liver and, despite its beneficial and therapeutic effects, increased circulating
FGF21 levels could be a prognostic indicator of metabolic imbalance and homeostatic disorders.
Preliminary data, obtained under the parental license, showed increased levels of circulating
hepatic FGF21 in Slc25a47hep-/- mice, indicating that the loss of this mitochondrial carrier is
triggering organismal compensatory responses. However, these compensatory responses could
represent a confounding factor when characterizing the specific role of SLC25A47 in the liver. In
order to overcome this issue, we plan to use a newly generated mouse line, which lacks both
FGF21 and SLC25A47 in the liver. In order to assess the metabolic implications of the double
deletion,8-12 weeks old mice (Fgf21hep-/- ; Slc25a47hep-/- DKO vs Fgf21hep-/- vs Slc25a47hep-
/- vs Fgf21hep+/+ ; Slc25a47hep+/+ mice) will undergo a non-invasive metabolic phenotyping as
described in section A2: we will check body composition (EchoMRI), energy expenditure (by
indirect calorimetry with Comprehensive Lab Monitoring System - CLAMS), food intake, feces
composition, glucose tolerance (by glucose tolerance test - GTT), insulin resistance (by insulin
tolerance test - ITT), pyruvate tolerance test and a non-lethal cold test. Finally, mice will be
sacrificed under overnight fasting (14h) after brief exposure to CO2 followed by cardiac puncture
for blood collection and organs harvesting. We will then perform both omics analyses (RNA
sequencing, proteomics, and metabolomics) and functional assays (such as oxygen consumption
assessments).
References: K. H. Kim and M. S. Lee, "FGF21 as a stress hormone: The roles of FGF21 in stress
adaptation and the treatment of metabolic disease," Diabetes Metab. J., vol. 38, no. 4, pp. 245-
251, 2014.
T. Inagaki, "Research Perspectives on the Regulation and Physiological Functions of FGF21 and
its Association with NAFLD," Front. Endocrinol. (Lausanne)., vol. 6, no. September, pp. 1-7, Sep.
2015.

Y. C. et al., "Activation of Liver FGF21 in hepatocarcinogenesis and during hepatic stress," BMC
Gastroenterol., vol. 13, no. 1, p. 67, 2013.
Study B: Elucidate the molecular pathways upstream and downstream of SLC25A47 in

hepatocytes (Figure 8). In this study, we will clarify the signalling events taking place upstream
and downstream of Slc25a47 in hepatocytes.
For this purpose, we will use primary hepatocytes. To derive primary hepatocytes, mice will be
anesthetized using a mix of Ketamine and Xylazine and liver will be perfused to harvest the
hepatocytes. We will use Slc25a47hep-/- mice and their respective controls (Slc25a47hep+/- and
Slc25a47hep+/+).
Duration of experimental series:
Study A1: Maximum total duration of 14 hours
Study A2: Maximum total duration of 10 weeks
Study A3: Maximum total duration of 5 weeks.
Study A4: Maximum total duration of 14 hours.
Study B: Mice will undergo a perfusion for hepatocytes harvesting. The perfusion time is about 20
min/mouse.
SC-32387-VD3221x1c - Last submitted to canton: 23.09.2021
New Amendment September 2021
In this amendment, we would like to ask for minor modifications of the previously proposed
studies. In particular, we would like to replace the taurine supplementation (in study A5) with
formate supplementation, as new molecular data revealed that SLC25A47 could be involved in
the hepatic choline and purine pathways, in which formate plays a key role.
We would also like to modify the duration of Aim A2 and Aim A7 (from 10 to 15 weeks) and add
a new non-invasive technique to precisely quantify the body fat (µCT, see section 25).

Additionally, given the importance of using both male and female animals, and in order to be
more representative of the human population, we would like to investigate the hepatic metabolic
differences of the two genders in our mouse model. So far, besides for primary hepatocyte
isolation, we only used male animals. Now, we would like to include female mice in the Aim A4
and study the possible sex-related variability in Slc25a47hep-/- mice.
Study A: Assess the function of Slc25a47 in hepatic metabolism
Study A1: unchanged
Update to Study A2 (new figure 2)
We would like to extend the HFHS diet study of 5 weeks (up to 15 weeks) to increase the clinical relevance
of our findings. Indeed, data from the literature show that, in order to see the first molecular and phenotypical
effects, the minimum amount of time for mice to be kept under obesogenic diet is 4 weeks. We therefore
decided to revise our HFHS study as shown in “new Figure 2”: mice will receive HFHS diet or CD for 4 weeks
prior starting the non-invasive phenotyping as described previously.
References:
Leonardo Recena Aydos, Luane Aparecida do Amaral, Roberta Serafim de Souza, Ana Cristina
Jacobowski, Elisvânia Freitas dos Santos, and Maria Lígia Rodrigues Macedo “Nonalcoholic Fatty
Liver Disease Induced by High-Fat Diet in C57bl/6 Models”. Nutrients 2019 Dec; 11(12): 3067.
Samina Bashira , Shakir Alia & Farah Khana “Partial Reversal of Obesity-Induced Insulin Resistance
Owing to AntiInflammatory Immunomodulatory Potential of Flaxseed Oil”. Immunological
Investigations 2015.
Study A3: unchanged
Update to Study A4 (new Figure 4)

We would like to implement our study and investigate the possible sex-related variability given by the loss of
SLC25A47 in mice. For this reason, we would like to include female animals in this study.

New Study A5 will include the same experiments and treatments described in the original Study A5; however,
we would like to replace the taurine supplementation with formate supplementation.
New data obtained under the license VD3221.1 linked SLC25A47 to the 1-carbon (1C) pathway and purine
synthesis in liver. In order to verify this hypothesis, we aim at supplementing the mouse diet with formate.
Formate is the simplest carboxylic acid that is normally present at low levels in blood and urine. It has
already been adopted to rescue animals with defects in 1C pathway enzymes (see references below). It is
harmless and very well tolerated by animals. Mice will be treated with 20-30 mg/ml sodium formate in the
drinking water) or normal water for 5-6 weeks.
References:
Jessica Momb, Jordan P. Lewandowski, Joshua D. Bryant, Rebecca Fitch, Deborah R. Surman, Steven
A. Vokes, and Dean R. Appling, “Deletion of Mthfd1l causes embryonic lethality and neural tube and
craniofacial defects in mice”, PNAS 2013; 549-554.
Yun Jin Pai, Kit-Yi Leung, Dawn Savery, Tim Hutchin, Helen Prunty, Simon Heales, Margaret E.
Brosnan,

John T. Brosnan, Andrew J. Copp& Nicholas D.E. Greene, “Glycine decarboxylase deficiency causes
neural tube defects and features of non-ketotic hyperglycinemia in mice”. Nat.Comm. 2015
Study A6: unchanged

In addition to what is already proposed, and with the aim of separating the role of SLC25A47 and FGF21 in
the context of hepatic pathological condition, we plan to challenge the liver by feeding the animals with a
high-fat high-sucrose (HFHS) diet for a maximal duration of 15 weeks. Fgf21hep-/- ; Slc25a47hep-/- DKO vs
Fgf21hep-/- vs Slc25a47hep-/- vs Fgf21hep+/+ ; Slc25a47hep+/+ mice will be tested for: body composition
(EchoMRI) and fat mass quantification (µCT), energy expenditure (by indirect calorimetry with
Comprehensive Lab Monitoring System - CLAMS), food intake, feces composition, glucose tolerance (by
glucose tolerance test - GTT), insulin resistance (by insulin tolerance test - ITT). Additionally, the mice will
undergo a pyruvate tolerance test and a non-lethal cold test, as described in Study A2.
Study B: unchanged
New Amendment May 2022
In this amendment, we would like to request minor modifications to the previously proposed
studies, and we would like to add four new mouse lines that will be critical in pursuing the
molecular and pathophysiological characterization of Slc25a47.
The first mouse line we are interested in is a whole-body knock-out of Slc25a47 (Slc25a47CMV-/-
= Slc25a47lox/loxCMVtg/0 - and controls Slc25a47CMV+/+ = Slc25a47lox/loxCMV0/0). The mouse line
we have been using so far presents a hepatocyte specific deletion of Slc25a47 (Slc25a47hep-/- =
Slc25a47lox/lox AlbCretg/0). However, when imaging liver samples from Slc25a47hep-/-, we found
patches of cells expressing Slc25a47. To understand their origin and their possible effects on the
pathophysiology, we would like to make use of a whole body knock out which cannot constitutively
express this protein in any cell type.
The second and third mouse lines we would like to study present a hepatocyte-specific
(indSlc25a47
hep-/- = Slc25a47lox/lox AlbCreERT2mut/+) and a whole-body (indSlc25a47CMV-/- = Slc25a47lox/lox

CAGGCreERTMtg/0) deletion of Slc25a47, induced upon tamoxifen administration. These mouse
lines allow to acutely delete Slc25a47 and study the very first events and pathways affected by
the absence of SLC25A47. These models permit to further investigate the transported solute, as
well.
The last mouse line we would like to add allows the isolation of purer and healthier mitochondria.
MITO-Tag mice present an HA tag on their mitochondria to allow clean and efficient isolation of
these organelles. Here, we crossed this line with Slc25a47hep-/-, to obtain efficient and pure
isolation of hepatocyte specific mitochondria. Studies conducted so far in our laboratory have

pointed to a central role of SLC25A47 in the control of mitochondrial fitness and function. Hence,
cleaner isolation is essential to finely understand SLC25A47 function in this organelle.
In molecular studies performed in cell lines, we have identified a phosphorylation site for
SLC25A47, which could regulate its function. To assess the importance of this phosphorylation
site in regulating SLC25A47 function in vivo, we would like to include in Study A6, where we
aimed to rescue the mouse’s phenotype by injection of an Slc25a47 construct into Slc25a47hep-/-
mice, a phosphorylation resistant and phosphorylation mimetic AAV8 vectors to analyse the
phosphorylation dependent effects of Slc25a47.
Under Study B, we would like to, first, add the new mouse lines for primary hepatocyte extraction
and include a new aim. We would like to assess biochemical changes at the cellular level in
primary hepatocytes from aged mice (Study B1). Preliminary studies in mice aged to 80 weeks
have shown the appearance of fibrosis and non-alcoholic steatohepatitis (NASH). To explain this
phenotype at the cellular level, how hepatocytes function is altered over time and the exact role of
Slc25a47 in fibrosis and NASH, we would like to extract hepatocytes from aged mice. We would
like to also include females, since the susceptibility to NASH is different between males and
females. Studying how the gender affects the phenotype may give further insights into Slc25a47
role in NASH development.
Update to Study A1 (Updated Figure 1)
Slc25a47 is a gene exclusively expressed in hepatocytes under physiological conditions. The

mouse model used thus far in this license (Slc25a47hep-/-) presents a constitutive hepatocyte
specific deletion of Slc25a47. We have performed RNA-scope studies using liver samples from
these mice and we have found patches of cells expressing Slc25a47. These cells should not be
hepatocytes since the deletion of Slc25a47 is Alb-Cre mediated. However, they could be either
hepatocytes that escape the Cre recombinase or other cell types which activate Slc25a47
expression to compensate for its absence in hepatocytes. Moreover, we cannot know if these
patches of cells are functional and if they are partially masking the phenotype we observed in the
Slc25a47hep-/- mice. Hence, we would like to study a new mouse model: a whole-body Slc25a47
knock-out model (Slc25a47CMV-/-). This model would clarify if the patches of cells represent a
different population of cells which activates expression of Slc25a47 and if indeed these cells are
influencing the phenotype. Using these mice, we would like to perform both omics analyses (RNA
sequencing, proteomics, metabolomics) and functional assays (such as oxygen consumption
assessments) in livers of Slc25a47CMV-/- and control mice (Slc25a47CMV+/+). 8 weeks old mice
will be euthanized after overnight fasting (14h) or in feeding condition (chow diet, SAFE 150, Safe
diets) after anaesthesia using a mix of Ketamine and Xylazine followed by cardiac puncture for
blood collection and organ harvesting.
New Study A1a (new Figure 1a)
We have indeed performed untargeted metabolomics studies using the Slc25a47hep-/- mice and
control liver tissue. These studies have shown that several metabolites involved in purine and
pyrimidine pathways are affected. However, we have used frozen samples for these studies. A

few central metabolites involved in purine and pyrimidine pathway are degraded during the
freezing steps. Hence, to further elucidate the role of Slc25a47 in this metabolic pathway, we
would need to perform targeted metabolomics. To obtain the maximum possible information, we
would need to prepare fresh tissue in specific buffers for metabolic assay (Tavazzi et al.) and we
would like to use the Slc25a47hep-/- and relative control mice (Slc25a47hep+/+). We chose to
use this line instead of the whole-body knock-out since we are interested in the hepatocyte
specific pathways which are affected by Slc25a47 deletion. Mice will be euthanized after overnight
fasting (14h) or in feeding condition (chow diet, SAFE 150, Safe diets) after anaesthesia using a
mix of Ketamine and Xylazine followed by cardiac puncture for blood collection and organ
harvesting.
Tavazzi, B., Lazzarino, G., Leone, P., Amorini, A. M., Bellia, F., Janson, C. G., ... & Giardina, B.
(2005). Simultaneous high performance liquid chromatographic separation of purines,
pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis
of inborn errors of metabolism. Clinical biochemistry, 38(11), 997-1008.
We have already performed non-invasive phenotyping of Slc25a47hep-/- and control mice

(Slc25a47hep+/+). However, when imaging liver samples from Slc25a47hep-/- mice using RNA-
scope, we observed patches of cells with expression of Slc25a47. Since Slc25a47 in this mouse
line is deleted only in hepatocytes, other cell types may activate Slc25a47 expression to
compensate for the phenotype. We do not know the origin of these cells and how their presence
affects the metabolic phenotype. Therefore, we would like to use a whole-body knock-out model
(Slc25a47CMV-/-) to clarify these questions. We would like to repeat the phenotypic analysis in
Slc25a47CMV-/-, whole body knock-out mice, and respective controls (Slc25a47CMV+/+). These
mice would present a constitutive deletion of Slc25a47 which is independent of albumin
expression. Therefore, they would allow us to verify that the observed phenotype is not affected or
partially masked by cells re-expressing Slc25a47. 8 weeks old Slc25a47CMV-/- and control
(Slc25a47CMV+/+) mice will be fed chow diet (SAFE 150, Safe diets) and will undergo a
phenotyping pipeline comprehensive of: body composition (EchoMRI, MicroCT), energy
expenditure (by indirect calorimetry with Comprehensive Lab Monitoring System - CLAMS), food
intake, feces composition, glucose tolerance (by glucose tolerance test - GTT), insulin resistance
(by insulin tolerance test - ITT). Additionally, the mice will undergo a pyruvate tolerance test and a
non-lethal cold test.
New Study A3a (New Figure 3a)
In order to study the acute effects of Slc25a47 deletion and to narrow down the pathways
regulated by Slc25a47; we would like to use two new Slc25a47 inducible knock-out models. The
use of these inducible models allows a more precise control of Slc25a47 expression in time and a
better study of the effects of its deletion in mice. Specifically, we would use tamoxifen inducible
Slc25a47 whole-body and hepatocyte specific knock-out models, indSlc25a47CMV-/- and

indSlc25a47hep-/- mice, respectively, with relative controls indSlc25a47CMV+/+ and

indSlc25a47hep+/+. Since Slc25a47 is specific to hepatocytes, its transient deletion in these cells
could highlight specific pathways and point to metabolites which are only affected when Slc25a47
is knocked-out in hepatocytes. However, we have observed recovery of expression in cell
patches, therefore the use of a whole-body knock-out would allow us to identify possible pathways
which are masked by the cells that recover Slc25a47 expression. The comparison of these two
models will help to identify the pathways which are due to hepatocyte dysfunction, and the ones
which may be linked to the patches of cells. Hence, we would like to use these two mouse lines
for both omics analyses (RNA sequencing, proteomics, metabolomics) and functional assays
(such as oxygen consumption assessments) in livers of indSlc25a47CMV-/- and indSlc25a47hep-
/-, and control mice (indSlc25a47CMV+/+ and indSlc25a47hep+/+). The deletion of Slc25a47 will
be induced at 8 weeks of age by intra-peritoneal (IP) injection of 100 μL tamoxifen (10 mg/mL
diluted in filtered sunflower oil) for 5 consecutive days (Lefort et al.). The control mice
(indSlc25a47CMV+/+ and Slc25a47hep+/+) will be treated by an IP injection of 100 μL of vehicle
(filtered sunflower oil) for 5 consecutive days. After the 5 days of injections, mice will be allowed to
rest for one week to recover from any stress or discomfort they may have experienced due to the
injections and to allow any residual oil to wash out. 10 weeks old mice will be euthanized after
overnight fasting (14h) or in feeding condition (chow diet, SAFE 150, Safe diets) after anaesthesia
using a mix of Ketamine and Xylazine followed by cardiac puncture for blood collection and organ
harvesting.
Lefort, C., Roumain, M., Van Hul, M., Rastelli, M., Manco, R., Leclercq, I., ... & Cani, P. D. (2020).
Hepatic NAPE-PLD is a key regulator of liver lipid metabolism. Cells, 9(5), 1247.
In Study A5, we would like to rescue the Slc25a47hep-/- phenotype by means of metabolite
supplementation. In the previously approved study, we were supplementing 3 weeks old
Slc25a47hep-/- and Slc25a47hep+/+ mice with 2% betaine, 2% choline or 2% formate (in the
drinking water) or normal water for 5-6 weeks. However, to obtain the maximal possible effect on
mice recovery, we would like to feed the mother with the metabolite in the water (Suiwala et al.).
This procedure should allow to prevent the phenotype development. Once the phenotype is
observed, it may become difficult if not impossible to reverse it. Hence, feeding the mother would
be more relevant as we are interested in understanding the transported metabolite and pinpoint
the pathways affected by Slc25a47 knock out, rather than a therapeutic effect of these
metabolites.
In details, experimental litters will be generated by overnight mating of one parent AlbCre*0 and
one parent AlbCre*Tg. The use of these parents allows to avoid the generation of heterozygote
mice. On detection of a copulation plug, the following morning, dams will be separated and
treated by feeding water containing 2% betaine, choline or formate and control (water only). Only
male mice will be included in the study. After weaning, male mice will still have access to water
supplemented with betaine, choline, formate or water only. Mice will be fed chow diet (SAFE 150,
Safe diets). At 8 weeks of age, mice will be fasted overnight (14h), and they will be euthanized

after anaesthesia using a mix of Ketamine and Xylazine followed by cardiac puncture for blood
collection and organ harvesting.
Sudiwala, S., De Castro, S. C., Leung, K. Y., Brosnan, J. T., Brosnan, M. E., Mills, K., ... &
Greene, N. D. (2016). Formate supplementation enhances folate-dependent nucleotide
biosynthesis and prevents spina bifida in a mouse model of folic acid-resistant neural tube
defects. Biochimie, 126, 63-70.
Molecular studies in cellular models have unravelled a phosphorylation site for SLC25A47, which
could represent a site of regulation of this protein. In order to fully characterize SLC25A47
regulation and validate the site of phosphorylation in vivo, we would like to use four constructs:
AAV8-Slc25a47, AAV8-Slc25a47 phospho-resistant, AAV8-Slc25a47 phospho-mimetic and
AAV8-Empty as a control. The use of these four constructs will allow us to observe whether
recovery of Slc25a47 expression can rescue the phenotype and how the phosphorylation site
influences Slc25a47 molecular signature and phenotype. Slc25a47hep-/- mice of 8-12 weeks of
age will be injected with the constructs, 5 weeks after injection mice will be euthanized after
anaesthesia using a mix of Ketamine and Xylazine followed by cardiac puncture for blood
collection and organ harvesting.
New Study A8 (new Figure 8)
SLC25A47 is a mitochondrial carrier specifically expressed in hepatocytes. Previous experiments,

performed in our laboratory under the license VD3221.1, have shown that the loss of function of
this protein can have detrimental effects on the hepatocytes’ mitochondria. We have analyzed
mitochondrial fraction contamination by other organelles and found that our mitochondria isolation
protocol is suboptimal. Hence, we would like to use MITO-Tag mice. MITO-Tag mice present a
HA-tag specific to mitochondria (Bayraktar et al.) and by crossing these mice with Slc25a47hep-/-
and controls (Slc25a47hep+/+) mice we would obtain purer, i.e. no contamination by other
organelles, and quicker, i.e. healthier organelle, isolation of hepatocyte specific mitochondria.
Hence, to further investigate the effects of Slc25a47 deletion in mitochondria, we would like to
extract them from Slc25a47MITOhep-/- and controls (Slc25a47MITOhep+/+). We would like to use
these mitochondria for different functional assays including respirometry assays and omics
analysis. 8-12 weeks old mice will be euthanized under overnight fasting (14h) or fed (CHOW diet)
condition after anaesthesia using a mix of Ketamine and Xylazine followed by cardiac puncture for
blood collection and organ harvesting.
Bayraktar, E. C., Baudrier, L., Özerdem, C., Lewis, C. A., Chan, S. H., Kunchok, T., ... & Chen, W.
W. (2019). MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria

from specific cell types in vivo. Proceedings of the National Academy of Sciences, 116(1), 303-
312.
Update to Study B: Investigate the repercussions of the loss of Slc25a47 at the cellular level
(Figure 9)
In order to analyse, at the cellular level, the pathways affected by loss of Slc25a47, we would like
to use primary hepatocytes. We previously used Slc25a47hep-/-, Slc25a47hep+/- and controls
Slc25a47hep+/+ for this analysis. We would like to also make use of hepatocytes extracted from
Slc25a47MITOhep-/- and Slc25a47MITOhep+/+ mice to continue the analysis of the effects of
Slc25a47 knock-out at the mitochondrial level using mitochondria extracted from primary
hepatocytes. Mitochondria isolated from MITOtag mice are cleaner, e.g. no contamination from
other organelles, and the isolation is more efficient, e.g. reduced loss of mitochondria and reduced
time of isolation which is linked to mitochondria health maintenance. We would also like to isolate
primary hepatocytes from indSlc25a47CMV-/- and indSlc25a47hep-/- and relative controls
(indSlc25a47CMV+/+ and indSlc25a47hep+/+) to analyse how the acute deletion of Slc25a47
affects the molecular phenotype in hepatocytes. This assay could help to identify the cellular
pathways directly affected by Slc25a47 deletion. Lastly, we would like to isolate primary
hepatocytes from Slc25a47CMV-/- and relative controls (Slc25a47CMV+/+). We would like to
include these cells in case masked phenotypes (compared to Slc25a47hep-/-) are observed
during metabolic phenotyping of these mice and further analyses at the cellular level are needed
to clarify the origin of these discrepancies. To derive primary hepatocytes, mice will be
anesthetized using a mix of Ketamine and Xylazine and liver will be perfused to harvest the
hepatocytes.
New Study B1: Slc25a47 biochemical and respirometry differences in primary hepatocytes from
aged mice (Figure 10)
Preliminary studies using aged mice, 80 weeks old, have shown a propensity for Slc25a47hep-/-
mice to develop fibrosis and non-alcoholic steatohepatitis (NASH). Since we would like to study
the molecular role of Slc25a47 in the development of this phenotype, we would like to extract
primary hepatocytes from aged Slc25a47 mice. Interestingly, NASH has been known to be
affected by gender difference (Matsushita et al.). In our preliminary studies, fibrosis and NASH
was present in both females and males. This phenotype could further be explored at the cellular
level to clarify which pathways are affected by Slc25a47 since they would need to be gender
independent. Hence, we would like to extract primary hepatocytes from both males and females of
young (8-12 weeks old) and aged (80 weeks old) mice. To analyze the differences between the
conditions, we would like to perform functional assays including respirometry assays and omics
analysis. To derive primary hepatocytes, mice will be anesthetized using a mix of Ketamine and
Xylazine and liver will be perfused to harvest the hepatocytes. We will use Slc25a47hep-/- mice and
their respective controls (Slc25a47hep+/+).

Matsushita, N., Hassanein, M. T., Martinez-Clemente, M., Lazaro, R., French, S. W., Xie, W., ... &
Tsukamoto, H. (2017). Gender difference in NASH susceptibility: roles of hepatocyte
Ikkβ and Sult1e1. PLoS One, 12(8), e0181052.
Mice of wrong age, gender or genotype
The technique we have been using so far for primary hepatocytes isolation, sample freezing, and
organ collection are suboptimal.
Only around 2 in 3 primary hepatocytes isolation is successful. However, the reason for the
unsuccess is unclear. Hence, we would like to use animals which are not fit for our experimental
protocols, i.e. wrong age, gender or genotype, to optimize the technique, e.g. different placing of
the clamp, of the needle, different speeds of perfusion, adding anticoagulation agents and others.
Moreover, the most difficult part of primary hepatocytes isolation protocol is the placing of the
needle. Hence, excess mice could be used by new personnel for training. We have also observed
that freezing techniques, we have used so far in the laboratory, are state of the art for some
techniques, yet metabolomics and analysis of more transient reactions may be affected by the
speed or freezing methods we have been implementing. Hence, we would like to use some of
these animals to study the best order of collection of organs and snap freezing method for these
more delicate assays.
New Pilot Study A9: Rescue of the Slc25a47 deletion by AAV8-mediated re-expression of
Slc25a47 and Wars (New Figure 11)
Slc25a47 hepatocyte specific knock-out mice have been recently published (Bresciani et al.,
2022). However, some concerns about the model were raised during revision. The raised concern
is about whether recombination of the loxP sites downregulates another important gene: Wars. To
verify this, we would like to perform a rescue experiment. For this purpose, we will acutely delete
Slc25a47 in Slc25a47lox/lox mice using AAV8 mediated Cre deletion. Specifically, we would like to
perform an acute deletion of Slc25a47 and concurrently re-express Slc25a47, Wars, or GFP
(control). Mice will receive tail vein injection of the indicated viruses at 8-weeks of age, 12 mice
per condition at matching viral load: AAV8_hAAT-Cre + AAV8_hAAT-GFP (control); AAV8_hAAT
-Cre + AAV8_hAAT -Slc25a47 (Slc25a47 rescue); and AAV8_hAAT -Cre + AAV8_hAAT -Wars
(Wars rescue). We chose to use the human alpha 1 antitrypsin (hAAT) promoter, since this is
hepatocyte specific, allowing us to target Cre, Slc25a47 and Wars to hepatocytes. Slc25a47hep-/-
have reduced body weight compared to Slc25a47hep+/+. Therefore, after injection, mice weight will
be measured every other day, to verify body weight after rescue. Two weeks after injection, mice
will be fasted overnight (14h), and euthanized after anesthesia using a mix of Ketamine and
Xylazine followed by cardiac puncture for blood collection and organ harvesting.

New Study A9: Analysis of the glucose phenotype after the rescue of the Slc25a47 deletion by
AAV8-mediated re-expression of Slc25a47 and Wars (New Figure 12)
Pilot Study A9 will allow us to identify the main driver of the phenotypes observed in the
Slc25a47hep-/- mice. However, it has recently been published that Slc25a47 is correlated with
gluconeogenesis (Jin-Seon Yook et al., 2022). Therefore, we would like to analyze if this
phenotype is indeed driven by Slc25a47. To this end, we would like to perform an acute deletion
of Slc25a47 and concurrently re-express Slc25a47, Wars, or GFP (control). Mice will be tail vein
injected at 8-weeks of age using AAV8 mediated viral vectors. 12 mice per condition will receive
matching viral load of AAV8_hAAT-Cre + AAV8_hAAT-GFP (control), AAV8_hAAT -Cre +
AAV8_hAAT -Slc25a47 (Slc25a47 rescue), and AAV8_hAAT -Cre + AAV8_hAAT -Wars (Wars
rescue). Two weeks after the injection, mice glucose phenotype will be analyzed by means of
glucose tolerance (by glucose tolerance test - GTT), insulin resistance (by insulin tolerance test -
ITT), and pyruvate tolerance (by pyruvate tolerance test – PTT) tests. The tests will be performed
every other week (GTT week 10, ITT week 12, and PTT week 14) to allow the mice to rest for
enough time. Mice weight will be recorded every week to monitor the body weight phenotype
observed in the Slc25a47hep-/-. Two weeks after the last test, mice will be fasted overnight (14h),
and euthanized after anesthesia using a mix of Ketamine and Xylazine followed by cardiac
puncture for blood collection and organ harvesting.
Yook, Jin-Seon, et al. "A liver-specific mitochondrial carrier that controls gluconeogenesis and energy
expenditure." bioRxiv (2022).
New Study A10: Phenotypic analysis of Slc25a47 silencing in-vivo by means of AAV8-shRNA
(New Figure 13)
Since the Slc25a47lox/lox model has been put into question, we would like to use a different model
to study Slc25a47 deletion. Specifically, we would like to use AAV8 mediated silencing, obtained
by using short-hairpin RNA (shRNA). Since this method is based on injection of AAV8-shRNA
particles in C57BL/6J mice, no DNA recombination issues should be present as the silencing will
be at the level of the RNA. Therefore, the use of these mice will allow us to study the effects of
Slc25a47 silencing without co-founding factors. To this end we will use 8 weeks old C57BL/6J
mice, that will receive tail-vein injection of either AAV8-shRNA GFP scramble (control, 12 mice) or
AAV8-shRNA Slc25a47 (12 mice). Since Slc25a47hep-/- have reduced body weight compared to
Slc25a47hep+/+, after the injection, mice weight will be measured every other day. Two weeks after
injection, mice will be fasted overnight (14h), and euthanized after anesthesia using a mix of
Ketamine and Xylazine followed by cardiac puncture for blood collection and organ harvesting.

Cunningham, R. P., Moore, M. P., Dashek, R. J., Meers, G. M., Takahashi, T., Sheldon, R. D., ... & Rector, R. S.
(2021). Critical role for hepatocyte-specific eNOS in NAFLD and NASH. Diabetes, 70(11), 2476-2491.
New Study A11: SLC25A47 maturation in vivo (New Figure 14)
Preliminary data indicates that SLC25A47 may be cleaved in hepatocytes to obtain a shorter
protein. However, in cellular models this modification is not observed. Therefore, we hypothesize
that the physiological environment of the liver is necessary to allow the protein to mature. To
validate this hypothesis, we would like to express Slc25a47-Flag tagged protein in vivo. Yet, we
do not have data to point whether the cleavage happens on the C- or N-terminus. Therefore, we
propose to express two Flag-tagged constructs: AAV8_hAAT-Slc25a47 N-terminus Flag and
AAV8_hAAT-Slc25a47 C-terminus Flag. Moreover, to validate the correct expression of the
constructs in hepatocytes, we will include a AAV8_hAAT-GFP as a control. We chose to use
human alpha 1 antitrypsin (hAAT) promoter, since this is hepatocyte specific. 8 weeks old
C57BL/6J mice will receive tail vein injection of the indicated constructs: AAV8_hAAT-Slc25a47
N-terminus Flag (12 mice); AAV8_hAAT-Slc25a47 C-terminus Flag (12 mice); and AAV8_hAAT-
GFP (control, 12 mice). Two weeks after injection, mice will be fasted overnight (14h), and
euthanized after anesthesia using a mix of Ketamine and Xylazine followed by cardiac puncture
for blood collection and organ harvesting.
Document(s): 3
Questions/ Answers
New Study A2 et A7 : Quel sera le poids supplémentaire pris par les souris pendant les 5 semaines additionnelles de diète
HFHS? Jusqu'où est-il raisonnable d'aller avec un modèle d'obésité?
Answered - 23.09.2021 10:38:29
Thank you for the question. Normally, mice under obesogenic diets- for 15 weeks- could reach ~45-50 g of body
weight. In the additional 5 weeks of experiment we expect approximately 7-10 g of body weight gain. This weight
is perfectly well tolerated by the animals, which can still behave normally in the cage (including feeding and
drinking without particular issues). Please note that researchers administer high fat high sucrose diet to mice up to
20 or 30 weeks (A. Neuhofer et al 2014; T.S.Mcmillen et al 2013). However, the goal of our experiments is not to
obtain the most obese mouse model, but to study the pathological repercussions of the loss of our proteins of
interest.
Le fait que la délétion spécifique de SLC25a47 dans les hépatocytes n'ait pas empêché totalement l'expression de
SLC25a47 dans la lignée que vous avez utilisée jusqu'ici ne rend-il pas caduque l'utilisation de lignées inductibles
"hépatocytes spécifiques"?

Questions/ Answers
Answered - 18.05.2022 08:48:56
Thank you for your question. We would like to take the opportunity to clarify some points. The Alb-CRE-driven
model that we have been using until now gave very satisfactory results with more than 90-95% deletion achieved
in the liver. A deletion of 90-95% was already sufficient to obtain a streaking phenotype. Moreover, Alb-CRE
represents the best model to date to obtain hepatocyte specific deletion of genes of interest. It is very interesting
that the AlbCre mediated chronic deletion of Slc25a47 lead to cells expressing Slc25a47 (in compensation
maybe). The other models we request will allow us understand these results and to study better our protein of
interest: the inducible deletion models represent a complement to study the pathways which are influenced by
Slc25a47 deletion first (since compensatory effects would take several days to weeks to come into play); whereas
the CMV-Cre is intended to understand the origin of the cells re-activating Slc25a47 expression; lastly the MITO-
Tag model is meant to permit a more accurate study of mitochondria in our model of interest.
Slc25a47_tm1c_AlbCRE represents for us, at the moment, the most characterized and defined model. If any of
the new mouse lines reveal themselves to be a better model for Slc25a47 study, we will use that one later on.
However, for the moment, since we are interested in SLC25A47 in hepatocytes’ health, the
Slc25a47_tm1c_AlbCRE is a very valid and useful model.
Study A5. Do you expect a disappearance of the phenotype (smaller size) in Slc25a47 ko when dams are treated with
feeding water? If so, can you use this parameter to compare feeding regimens?
Answered - 30.06.2022 12:06:02
Thank you for your question. Indeed, we expect the treated Slc25a47 KO mice to weight similarly to the Slc25a47
wild-type mice. Monitoring the body weight of the mice would indeed be a good indication of the treatment effects.
We have included mice weight monitoring in the new scheme, Revised Updated Figure 5. Mice will be weighted
during tagging and once a week after weaning. To reduce the stress, mice will be tagged by trained personnel in
UDP (please find here below the list of personnel) and weighted during tagging. Thereafter, mice weight will be
collected once a week, after weaning, by personnel in Auwerx and Schoonjans group. As we cannot modify
section 11, please find below the list of the UDP personnel who can tag and weight the mice: Maude Zahno,
Mohamed Bouaziz, Nobs Véronique, Dalil Ait-Bara, Giacomo Diaceri, Xavier Quaglia, Céline Waldvogel and Roy
Combe.
Pourquoi l'intervalle entre le dernier test et le sacrifice est-il désormais de 3 semaines (vs 2 dans la demande mère)?
Answered - 23.09.2021 10:38:29
Thank you for noticing the change. We would like to give one extra week of recovery for the mice that underwent
the non-invasive phenotyping, in order to be able to euthanize them under the best conditions for metabolic
studies (i.e the less “stressed” as possible).
Y a-t-il actuellement des souris de l'étude A2 en expérience?

Answered - 23.09.2021 10:38:29
No, we do not have any mice for Study A2 currently under experiments.
When using the inducible whole KO, how long before sacrifice will procedures on these animals last? The long phenotyping
procedure must be preceeded and justified by evidence of absence of Slc25a47cells.
Answered - 30.06.2022 12:06:02
Thank you for your question. Slc25a47 inducible whole-body knock-out will not undergo an extensive phenotyping
pipeline. This pipeline is reserved for the whole body knock out model (Slc25a47_tm1c_CMVCre), which is not
inducible. Before performing any phenotyping assays on the inducible whole-body knock-out, we will completely
validate the mouse line. This validation will be performed using the mice allocated to respirometry assay.
Respirometry assay allow us to understand whether Slc25a47 has been knock out. Moreover, we measure body
weight and collect organs after euthanasia. This implies that the Biobanked samples can be used to validate the
line by qPCR, WB and RNAscope
Do you have evidence that the CMV promoter will lead to a less patchy expression than the Alb promoter in the liver? It may
worth to verify complete absence of Slc25a47: have you performed this analysis?

Questions/ Answers
Answered - 30.06.2022 12:06:02
Thank you for your question. The CMV promoter is a ubiquitous promoter, as opposed to the hepatocyte-specific
albumin promoter. Because of its constitutive nature, the CMV promoter is active very early during embryogenesis
(doi.org/10.2144/000113270) and will recombine the loxP site (and thus excise Slc25a47) before germ-layer
specification. In this configuration, no cell can escape recombination or reactivate expression as they will all derive
from recombined embryonic stem cells. That being said, we will validate the mouse line before performing the
phenotyping pipeline and in particular perform RNAscope on liver samples to validate absence of patches.
New Study A2 et A7: Vos nouveaux schémas expérimentaux indiquent un test de tolérance au pyruvate qui ne figure pas
dans les schémas de la demande mère. Or, ce test semble prolonger la durée de l'expérience de 3 semaines. À quel
moment aviez-vous prévu de le réaliser pour la demande mère?
Answered - 23.09.2021 10:38:29
Thank you for noticing the change. Indeed, pyruvate tolerance test was correctly approved in the parental license
VD3221.1, however we realized that the test was never added to the timeline file. We apologize for this mistake.
This is one of the reasons why we decided to amend the Study A2 and A7, so we could be able to perform all the
phenotyping tests both in control (CD) and HFHS diets.
Study A5 : Pourquoi choisissez-vous de remplacer le groupe taurine plutôt que le groupe bétaïne ou choline?
Answered - 23.09.2021 10:38:29
We decided to replace the taurine because of our new data concerning SLC25A47 being part of the hepatic
choline oxidation pathway. This pathway includes many metabolites important for mitochondrial health and liver
physiology (it is, indeed, a largely liver-specific pathway). Betaine, choline and formate are all part of this pathway,
while taurine is not. We would like to focus our rescue study on our new- and more promising- hypothesis.
Therefore, we decided to exclude taurine as a candidate as we are not convinced of its efficacy, in our model,
anymore. Replacing taurine with formate will also allow us to not increase the number of animals required for this
experiment, since we will not need additional mice.
Study A6 - Dans le paragraphe consacré à l'étude A5, vous écrivez "Once the phenotype is observed, it may become
difficult if not impossible to reverse it". Qu'est-ce qui vous permet de penser que ça ne sera pas le cas avec le protocole
d'injection d'AAV?
Answered - 18.05.2022 08:48:56
Thank you for your question. In Study A5, we are using metabolites supplementation. The reason we would like to
start the treatment, as soon as possible, is that delaying the metabolite supplementation can induce pathways
different from the physiological one to compensate for the phenotype. Hence, it may be that once these pathways
are activated, the proposed metabolite is not part or has a different role in these pathways. This difference
translates into an inability for the metabolite to rescue the phenotype. The approach in Study A6 is different. By
using AAV8 injection, we re-activate the gene expression. Therefore, once the protein is again present, the
original pathway should re-activate/function properly.
Pourriez-vous indiquer la séquence chronologique, ces prochains mois, des différentes expériences de cette demande
complémentaire?
Answered - 18.05.2022 08:48:56
Please find the timeline here below. We have mice which do not fit the criteria for other experiments (age) that we
are allowing to age to 80 weeks to euthanise them under Study B1 before the end of this license. This method
allows us to obtain cellular results using primary hepatocytes in aged mice, without breeding more animals.
New Study A2 et A7: pourquoi le test de tolérance au pyruvate et le test d'exposition au froid sont-ils réalisés selon une
chronologie inverse dans vos deux études?

Questions/ Answers
Answered - 23.09.2021 10:38:29
Thank you for noticing the mistake. The order of the two last tests is not really crucial for us, however we would
like to perform the cold test first and the pyruvate tolerance test as last experiment. We uploaded a corrected
version of New Figure A7 in a question below.
Avez-vous observé un résultat, ne serait-ce que partiel, des différentes supplémentations effectuées dans le cadre de l'étude
A5? Ou n'avez-vous pas effectué cette étude à ce stade?
Answered - 18.05.2022 08:48:56
Thank you for your question. We have not started this study yet.
Dans l'étude A1, n'y a-t-il aucune possibilité de diminuer le nombre d'animaux? Aucune succession chronologique qui
permettrait de renoncer à des expériences peu fructueuses par exemple?
Answered - 24.04.2020 09:48:55
All the mice will be euthanized for different purposes (omics analyses and functional assays), which are
independent experiments that address independent biological questions. Therefore, considering the importance,
for our study, of the nutritional status (fed or fasted) and the necessity to keep all the appropriate controls, we
cannot reduce the number of animals any further without jeopardizing the outcomes/conclusions.
24 Preparation of animals for the experiment
Assessment of the preparation of animals for the experiment (art. 119 Animal Welfare Ordinance (SR 455.1)): Description of screening
examination,preparation to the experimental conditions, method of marking or identification and combination with the method of
genotyping.
- All animals are weaned and marked by taking an ear biopsy at the age of 3 weeks. The biopsy is used for
genotyping. Animals that will be enrolled in the experiment at the age of 0d (study A4) will be genotyped using
qPCR.
- Animals are adapted to the animal house environment at least for one week before starting the
study.
All animals are weaned and marked by toe clipping between day 4 and 12 after birth. The biopsy
is used for genotyping.
Document(s): 0
25 Procedures / manipulations on the animal
Assessment of licence conditions (art. 140 Animal Welfare Ordinance (SR 455.1)) and suitability(section 38) of the method (art. 137
para. 3 Animal Welfare Ordinance (SR 455.1): Indicate the details of the various manipulations/interventions on the animal.
Study A1 and A4 - Fasting protocol: mice will be fasted for 14 hours (from 6 pm to 9 am the day after) and
then euthanized by brief exposure to CO2 (Figure 1 and 4).

Study A2 - High-fat, high-sucrose diet: Mice will receive HFHS diet (Harlan Laboratories,
TD.08811) in comparison with normal chow diet (Figure 2).
Study A3 and A6 - AAV8 injection: mice will be injected through the tail vein with AAV8 viral
particles (150 micro-liters saline). We will use a titer of 1.0x1012 vg viral particles per injection that
we already tested and optimized previously in the lab (Figure 3 and 6).
Perfusion: Anesthetized mice will be perfused at the portal vein with 70 ml of iso-osmotic medium
without calcium, followed by 55 ml a medium containing calcium and Liberase Blendzyme (Roche)
required for the dissociation of hepatocytes for approximately 5 minutes. The liver is then
dissected and hepatocytes are collected for ex vivo studies.
Non-invasive metabolic phenotyping: all the tests described below have been validated by a
consortium of European laboratories (EUMORPHIA). The consortium has proposed standard
procedures for a variety of tests for phenotyping mice (European Mouse Phenotyping Resource of
Standardised Screens (EMPResSS, http://empress.har.mrc.ac.uk). These tests are standard and
considered appropriate means for phenotyping mouse.
The procedures 1 to 4, as described below, were already approved in the parental license
VD3221.
1. Body composition by EchoMRI
1.a. Purpose This test measures the body composition using EchoMRI (Echo Magnetic
Resonance Imaging). The percentages of body fat and lean mass will be measured in awaken
mice.
1.b. Experimental technique Before each test, the system must be calibrated using a known
standard provided by the manufacturer Echo Medical System. The weight of mice is written down
and each animal is placed in a tube of the appropriate size which is then placed in the machine.
The mouse is subjected to a predetermined sequence of radio frequency energy and the software
saves EchoMRI spectra corresponding to each mouse over a period of 47 seconds. Three
independent sequential scans are performed for each mouse and data are automatically
transferred to a database. This technique gives access to the lean tissue mass, fat mass and
body fluids, provided in grams. The measurement takes 5 minutes per animal. At the end of the
test, the animal is returned to its cage.
1.c. Estimation of pain DG: Level 0 Stress : Low. The measuring tubes are narrow, but the
measurement lasts for only 1 minute.
1.d. References Tinsley, FC, Taicher, GZ, Heiman, ML. (2004) Evaluation of a new Quantitative
Magnetic Resonance (QMR) method for whole body composition analysis of souris. Obesity
Research. 12:150-160. Champy, MF, Argmann, CA, Chambon, P, Auwerx, J. (2006) Exploration
of metabolic and endocrine function in the souris. (Chapter 5) In de Angelis, HM., Chambon, P.,
Brown, S. (eds.) Standards of souris model phenotyping. Wiley-VCH, Weinheim.

2 Measure energy expenditure of animals by indirect calorimetry (CLAMS)
2.a. Purpose Measurement of energy expenditure of mice by indirect calorimetry (measuring

oxygen consumption by the system CLAMS). The spontaneous locomotor activity and ingestion of
food and water are simultaneously recorded.
2.b. Experimental technique The system consists of an open circuit for incoming and outgoing air
in closed plastic cages (1 mouse per cage, 206.25 cm2 surface areas, 12.7 cm height). Very
precise detectors measure the O2 and CO2 from the air entering and leaving (the flow of air into
the cages is constant) so that an assessment rate of oxygen consumed during a given period is
obtained. Infrared detectors, at 2 and 8.5 cm of the bottom of the cage, allow recording locomotor
activity. Feeding behavior is measured automatically over several hours by evaluating the number
of food pellets distributed and the number of licks from two bottles. Food distribution is controlled
by a system with a photosensitive cell, which issues a new 20 mg pellet if the previous one was
removed. - Calibrate before the device (O2, CO2, food and water). - Place each mouse
individually in the appropriate plexiglass boxes. It can be placed 12 mice at a time. The animals
have access to food and water during the test. The animals are around 24 hours in the boxes. At
regular intervals, the camera makes automatic measurements of O2 and CO2 in and out of each
box, and measures the intake of food and drink automatically. - The spontaneous activity is also
automatically measured throughout the test (24 hours). The test can be repeated over several
days as part of a study to analyze the behavior of adaptation to a new environment. - At the end of
the test, animals are placed in their cage with food and water.
2.c. Estimation of pain
DG: Level 1 Stress : possible stress relatif au changement d'acclimatation (cages sans sciure et
différent feeder de nourriture)
2.d. References Heinrichs S.C. (2001). Souris feeding behavior: ethology, regulatory mechanisms
and utility for mutant phenotyping. Behav. Brain Res. 125: 81-88. Kopp C. (2001). Locomotor
activity rhythm in inbred strains of souris: implications for behavioural studies. Behav. Brain Res.
125: 93-96. Ballard T.M., Pauly-Evers M., Higgins G.A., Ouagazzal A.M., Mutel V., Borroni E.,
Kemp J.A., Bliethmann , Kew J.C. (2002). Severe impairment of NMDA receptor function through
targeted point mutation of the glycine binding site in souris leads to drug resistant, non-habituation
hyperactivity. J. Neurosci. 22 : 6713-6723.
3. Glucose tolerance tests (GTT : glucose tolerance test)
3.a. Purpose This test explores glucose metabolism over time. Hyperglycemia is caused by oral
administration of glucose; glucose is then measured in the blood over a three hours period. Oral
administration of glucose produces a more physiological glycemic shock, and allows for an
assessment of the contribution of gastrointestinal hormones to the secretion of insulin.
3.b. Experimental technique - The morning of the test day, each mouse is placed in individual
cages (without food and with a bottle of water). - The amount of glucose solution administered is
calculated according to the body weight of each animal: 2g glucose / kg of the animal. - The
mouse is then placed in a restraining cage to make an incision on the tail vein at the side using a

scalpel. - A drop of blood (20 microL) is taken to measure glucose concentration. Note the initial
rate of glucose (T0). - The wound is compressed a few seconds. - The glucose solution is then
administered orally by gavage. This step marks the beginning of timing. The mouse is then placed
back in its cage. - After 15 minutes the mouse is again placed in the restraining cage to collect
another drop of blood to measure blood glucose. Wherever possible, blood is collected from the
same incision by removing the clot with a paper towel and massaging the tail if necessary to bleed
again. Note the rate of glucose measured with the strip (T15) - Similarly, blood glucose was
measured at T30, T45, T60, T90, T120, T150 and T180 if blood glucose is not returned to its
basal levels (total blood volume taken is 9 x 20 = 180 microL). -20 extra microL will be collected at
T0, T15, T30, T60 to check insulin - The mouse is placed in its home cage with food and water.
3.c. Estimation of pain DG: Level 1 Stress : Stress due to the intraperitoneal injection of glucose
and collection of blood samples from the tail vein for glucose measurement.
3.d. References Montgomery, L.B. and Loria, R.M., and Coleman, P.H. (1988) Effects of Standard
Laboratory Souris Diets on expression of Diabetes in the db+/db+ diabetic mutant souris. Nutrition
Research 8 : 295-307. Loria R.M., Montgomery, L.B., Chinchilli V.M. (1990) Evaluation of Virus
Neutralisation Antibodies Levels in Diabetics Mutant Souris. J. Virological Methods 28 :235-244.
4. Insulin Tolerance tests (ITT)
4.a. Purpose Insulin, a glucose lowering hormone, is injected intraperitoneally and blood glucose
is monitored over time for 120 minutes. This test assesses the sensitivity to insulin.
4.b Experimental technique - The morning of the test day, each mouse is placed in individual
cages (without food and with a bottle of water) for 6 hours of fasting. - The amount of insulin
solution to be injected is calculated based on body weight: 0.5 IU insulin / kg of the animal. - The
mouse is then placed in a restraining cage for a small incision on the tail at the vein using a
scalpel. - A drop of blood (20 microL) is taken and deposited on a strip measuring blood glucose.
Note the initial rate of glucose (T0). - The wound is compressed a few seconds to stop the
bleeding. - The insulin solution was injected intraperitoneally, using a syringe. Start timing. The
mouse is placed in its cage. - After 15 minutes the mouse is placed in the restraining cage and
another drop of blood is taken to measure blood glucose. Wherever possible, blood is collected at
the same incision by removing the clot with a paper towel and massaging the tail if necessary to
make it bleed. Note the level of glucose measured with the strip (T15). - Similarly, measure blood
glucose at T30, T45, T60, T90, and T120 if blood glucose is not returned to its original level (total
blood volume taken is 7 x 20 = 140 microL). - The mouse is placed in its home cage with food and
water.
4.c. Estimation of pain DG: Level 1 Stress: Stress due to the intraperitoneal injection of insulin and
collection of blood samples from the tail vein for glucose measurement.
4.d. Reference Surwit, R.S., M.F. Seldin, C.M. Kuhn, C. Cochrane, M.N. Feinglos. (1991) Control
of expression of insulin resistance and hyperglycemia by different factors in diabetic C57BL6/6J
souris. Diabetes 40: 82-87.Hoboken, NJ.

5 Pyruvate tolerance test
5.a. Purpose This test is a variant of the OGTT, in which pyruvate is injected instead of glucose.
The pyruvate bolus elicits a glycemic excursion that reflects hepatic gluconeogenesis. Hepatic
gluconeogenesis is affected by metabolic diseases and regulates glucose homeostasis.
5.b, Experimental technique - The morning of the test day, each mouse, previously fasted for 6h,
is placed in individual cages (without food and with a bottle of water). - The amount of pyruvate
solution administered is calculated according to the body weight of each animal: 2g pyruvate/ kg
of the animal. - The mouse is then placed in a restrainer to make an incision on the tail vein at the
side using a scalpel. - A drop of blood (20 microL) is taken to measure glucose concentration.
Note the initial rate of glucose (T0). - The wound is compressed a few seconds. - The pyruvate
solution is then injected intraperitoneally. This step marks the beginning of timing. The mouse is
then placed back in its cage. - After 15 minutes the mouse is again placed in the restraining cage
to collect another drop of blood to measure blood glucose. Wherever possible, blood is collected
from the same incision by removing the clot with a paper towel and massaging the tail if
necessary to bleed again. Note the rate of glucose measured with the strip (T15) - Similarly, blood
glucose is measured at T30, T45, T60, T90, T120, T150 and T180 if blood glucose is not returned
to its basal levels (total blood volume taken is 9 x 20 = 180 microL). - The mouse is placed in its
home cage with food and water.
5.c. Estimation of pain DG: Level 1 Stress : Stress due to the intraperitoneal injection and
collection of blood samples from the tail vein for glucose measurement.
5.d Reference Alicia H. Clementi, Allison M. Gaudy,Teresa A. Zimmers, Leonidas G. Koniaris, and
Robert A. Mooney Deletion of interleukin-6 improves pyruvate tolerance in the leptin receptor-
deficient mouse. Metabolism. 2011 Nov; 60(11): 1610-1619. Virtu Calabuig-Navarro, Jun
Yamauchi, Sojin Lee , Ting Zhang , Yun-Zi Liu, Kelsey Sadlek , Gina M. Goudriet , Jon D.
Piganelli , Chun-Lei Jiang , Rita Miller , Mark Lowe , Hideyoshi Harashima and H. Henry Dong
FoxO6 Depletion Attenuates Hepatic Gluconeogenesis and Protects Against Fat-Induced Glucose
Disorder in Mice J. Biol. Chem. 2015, 290:15581-15594
6 Non-lethal cold test
6.a. Purpose The goal of this test is to determine the thermogenic capacities of mice which are
tightly linked to their metabolic flexibility.
6.b, Experimental technique Mice are placed in individual cages without food and bedding but with
access to water. The initial body temperature is measured by inserting a thermometer into the
rectum of the animal before placing the mice at 6°C Mice are then placed in a cold room at 6°C.
Body temperature (rectal measurements with a rectal thermometer and KY lubricant) is measured
every hour over a period of 6 hours. At the end of the experiment, mice will be anesthetized by
isoflurane inhalation, sacrificed by heart puncture. Following cardiac puncture, we will perform
decapitation and different organs involved in whole body energy expenditure will be collected.
6.c. Estimation of pain DG: 2
5.d Reference License VD3187 and VD3419

Amendement Septembre 2021

We would like to add a new procedure:
6 Adipose tissue quantification- µCT

6.a. Purpose
X-ray computed tomography is a medical imaging procedure that uses computer-processed X-rays to
produce tomographic images or 'slices' of specific areas of the body. These cross-sectional images are used
for diagnostic and therapeutic purposes in various medical disciplines.
6.b. Experimental technique
Adipose tissue segmentation between subcutaneous and visceral adipose tissue and their respective
quantification are determined using Quantum Fx CT scan (Perkin Elmer, Massachussets, USA) in
anesthetized mice with isoflurane (1.5-3%) during approx. 5 minutes for X ray micro computed tomography.
Adipose tissue quantification is assessed using a single field of view of 30mm with isotropic resolution of 59
um. This FOV would deliver an approximate dose of radiation exposure to 18 mGy which is a very low and
safe dose of Xray exposure and has no impact on mouse physiology.
Isoflurane is the preferred choice for anesthesia due to its quick reversible character and rather harmless
nature. The micro-CT procedures are not invasive and do not create any harm, therefore no analgesia is
required.
6.c. Severity Grade
Grade 1
New Study A7– High-fat, high-sucrose diet: Mice will receive HFHS diet (Harlan Laboratories, TD.08811)
in comparison with normal chow diet (New Figure 7).
Mouse caudal lateral vein injection
a. Goal
Administrate a solution intravenously in conscious mice.
b. Procedure
Mice have two caudal lateral veins and both are superficial and visible as dark lines along each
side of the tail. To induce vasodilatation and therefore make the veins more prominent, place the
mice under a heating lamp (not too close to avoid burning) for about 5 minutes before injection.
Restrain the mouse in a restrainer and hold the tail, rotate it slightly to see one of the lateral veins.
Disinfect the injection site with 70% ethanol and insert the needle (27 to 30 gauge) with a slight
angle. Inject slowly and be sure that the vein changes from dark to light. If this is not visible, this
means that the needle is not correctly positioned in the vein and that the procedure needs to be
repeated in a more distal part of the tail. Upon injection completion, remove the needle and apply
a compress on the injection site to avoid bleeding.

c. Severity Grade
Grade 1
d. Reference http://www.bu.edu/animalcare/procedures/injection-techniques/intravenous/
New Pilot Study A9, New Study A9 and A11 - AAV8 injection: mice will be injected through the
tail vein with AAV8 viral particles (150 micro-liters saline). Prior to injection, mice will be placed
under an infrared lamp to allow tail vein dilatation. Mice will be placed into the restrainer and the
injection site on the tail will be cleaned using EtOH 70% solution. A 1ml insulin syringes (BD
Micro-Fine+ Seringue Insuline 1ml 29Gx12,7mm) will be used to inject a titer of 1.0x1012 vg viral
particles, this concentration was previously tested and optimized in the lab (Figure 11, 12 and 14).
New Study A10 - AAV8 injection: mice will be injected through the tail vein with AAV8 viral
particles (150 micro-liters saline). Prior to injection, mice will be placed under an infrared lamp to
allow tail vein dilatation. Mice will be placed into the restrainer and the injection site on the tail will
be cleaned using EtOH 70% solution. A 1ml insulin syringes (BD Micro-Fine+ Seringue Insuline
1ml 29Gx12,7mm) will be used to inject a titer of 1.0x1011 vg viral particles, this concentration was
chosen as it is the one reported in literature for shRNA (Figure 13).
Cunningham, R. P., Moore, M. P., Dashek, R. J., Meers, G. M., Takahashi, T., Sheldon, R. D., ... & Rector, R. S.
(2021). Critical role for hepatocyte-specific eNOS in NAFLD and NASH. Diabetes, 70(11), 2476-2491.
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Questions/ Answers
Vous écrivez: "Inject slowly and be sure that the vein changes from dark to light. If this is not visible, this means that the
needle is not correctly positioned in the vein and that the procedure needs to be repeated in a more distal part of the
tail". Il me semble qu'en cas d'échec, le nouveau site d'injection devrait se situer plus haut sur la queue (plus proximal).
Pourriez-vous vérifier?
Il me semble que vous avez ajouté un microCT à votre pipeline de phénotypage. Pourriez-vous décrire la procédure et la
justifer dans le contexte de ce protocole?

Questions/ Answers
Answered - 18.05.2022 08:48:56
Thank you for your question. In the amendment VD3221.1c, we asked for a second method to analyse the
adipose tissue composition. This method has been approved then and we intend to use it for these new
experiments when the results from EchoMRI are not conclusive. We prefer EchoMRI whenever possible because
it results in less stress to the mice (no anesthesia needed).
In the Procedures / manipulations on the animal section under amendment September 2021 you may find the
description of the procedure. We also report it below:
6 Adipose tissue quantification- μCT
6.a. Purpose
X-ray computed tomography is a medical imaging procedure that uses computer-processed X-rays to produce
tomographic images or 'slices' of specific areas of the body. These cross-sectional images are used for diagnostic
and therapeutic purposes in various medical disciplines.
6.b. Experimental technique
Adipose tissue segmentation between subcutaneous and visceral adipose tissue and their respective
quantification are determined using Quantum Fx CT scan (Perkin Elmer, Massachussets, USA) in anesthetized
mice with isoflurane (1.5-3%) during approx. 5 minutes for X ray micro computed tomography. Adipose tissue
quantification is assessed using a single field of view of 30mm with isotropic resolution of 59 um. This FOV would
deliver an approximate dose of radiation exposure to 18 mGy which is a very low and safe dose of Xray exposure
and has no impact on mouse physiology.
Isoflurane is the preferred choice for anesthesia due to its quick reversible character and rather harmless nature.
The micro-CT procedures are not invasive and do not create any harm, therefore no analgesia is required.
6.c. Severity Grade
Grade 1
Concernant le point "2 Measure energy expenditure of animals by indirect calorimetry (CLAMS)", si je comprends
bien, il serait fait usage d'ancienne cage en forme de "T" qui se trouvent à l'EPFL.
Pourquoi ne pas faire usage des nouveaux modèles qui sont plus grands et permettent aux souris d'être maintenue sur de la
sciure, ce qui limiterait leur stress ?
Answered - 30.06.2022 12:06:02
Thank you for your question. Promethion offers a less stressful environment for the mice, and we do prefer it when
available. However, Promethion is very heavily booked, therefore we chose to include CLAMS to ensure
availability of one of the systems at the time indicated for phenotyping. Whenever possible, we will prefer the use
of Promethion. Of note, the Promethion system is fully booked until the end of Q1 2023.
Concernant les points "3. Glucose tolerance tests (GTT : glucose tolerance test)", "4. Insulin Tolerance tests
(ITT)" et "5 Pyruvate tolerance test", est-ce que 9, respectivement 7 et 9 prélèvements sanguins et un
gavage ne sont pas plutôt un degré 2 de contrainte en raison du cumul des procédures en un temps relativement court ?
Answered - 30.06.2022 12:06:02
Thank you for your question. Blood collection is performed only at 4 time points, at the other time points only a
drop of blood is needed to measure glycemia. It is indeed true that repetitive blood collection and a gavage can be
somewhat stressful for the mice. However, these assays are performed by trained personnel, that can handle the
mice with care to reduce to the minimum the stress. Moreover, the mice are allowed at least one week rest
between two phenotyping tests. Therefore, given the training, the few times blood is collected, and the resting
time, we believe this assay should be considered degree 1 which is in agreement with the recommendation of the
technical information 1.04 on severity degrees.
3. Test de tolérance au glucose: il y divergence entre deux paragraphes - solution de glucose administrée par gavage et
injection intrapéritonéale de glucose. Merci d'harmoniser 5. Test de tolérance au pyruvate: vous écrivez qu'il s'agit d'une
variante au OGTT, soit, sauf erreur de ma part, oral glucose tolerance test. Or, le pyruvate est administré i.p. Merci de
vérifier.Pourquoi faut-il contrôler l'insuline lors du test de tolérance au glucose et pas lors du test de tolérance au pyruvate?

Questions/ Answers
Answered - 24.04.2020 09:48:55
3. Thank you for bringing up this error. Indeed, there is a mistake in the text; glucose is administered only orally
via gavage and not by intraperitoneal injection. 5.-Pyruvate is normally administered via intraperitoneal injection,
while glucose could be administered either by i.p injection or gavage. We decided to give glucose because
injecting it in i.p would bypass the role of the gut in glucose metabolism. We want to remain as close as possible
from the physiological situation. We thus decided to perform a gavage administration. This will be done by an
experienced researcher so as to limit the stress on the animal. -Glucose homeostasis is tightly controlled by the
interplay of various hormones and organs. In the fasted state, glucose levels are prevented from dropping too low
(hypoglycemia) through two hepatic processes: glycogenolysis, the degradation of glycogen and
gluconeogenesis, the generation of glucose from non-carbohydrate carbon substrates including pyruvate and
lactate (produced by anaerobic metabolism of glucose in muscles). The classic interpretation of the pyruvate
tolerance test is that the change in glucose, in response to pyruvate, reflects hepatic gluconeogenesis. In this
context, there is no need of controlling the levels of insulin.On the contrary, administration of glucose (which is
comparable to a "postprandial" state) directly influences insulin levels. Thus, we decide to check
insulin during the OGTT.ReferenceHughey et al. "Approach to Assessing Determinants of Glucose
Homeostasis in the Conscious Mouse". Mamm Genome. 2014
26 Anaesthesia and / or analgesia
Assessment of the application in accordance with the implementation provisions (art. 135 Animal Welfare Ordinance (SR 455.1):
Indicate the medications to be used, the dose and volume, the intended routes of administrationand consider the need of continuing
administration.
For anesthesia we use a single injection of Xylazine 5-10 mg / kg IP plus Ketamine 80-100 mg / kg i.p.
Amendement September 2021

For anesthesia during the µCT technique we will use inhalation of isoflurane (4% in oxygen, 1L/min for
induction) and maintained under anesthesia mask (1-1.5% isoflurane, 1L / minute)
Rationale of anaesthesia / analgesia
Reasons for selecting or for not using analgesics and anaesthesia should be stated.
A mixture Xylazine / Ketamine is used for analgesia and anesthesia of mice for 20 to 30 minutes. This allows the
insertion of a catheter for the infusion of hepatocytes (Study B).

For the µCT technique, mice will be anesthetized by isoflurane as described above. We chose isoflurane
because it does not have as many effects on glucose metabolism and cardiac function as
ketamine/xylazine. Moreover, isoflurane is the most appropriate method for anesthesia during imaging, and
is also well tolerated by the mice.
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27 Methods of euthanasia
Assessment of the application concerning the proper conduct of euthanasia (art. 135 Animal Welfare Ordinance (SR 455.1)): Indicate
the method of euthanasia, stating the substance used, the dose and route of administration, and the procedure to ensure the death of
the animal.
Copy/Paste: Mice will be euthanised after a brief exposure to CO2 followed by cardiac puncture.
For perfusion, injectable anesthetic (ketamine & xylazine) will be used followed by bleeding.
As requested and indicated in the answer to the question. CO2 euthanasia will not be used
anymore. We prefer the use of an ip injection of a mix of ketamine 5-10mg/kg/xylazine 80-
100mg/kg followed by cardiac puncture (which is a method already described and approved in the
parental license).
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Questions/ Answers
Renoncez-vous au CO2 pour toutes les euthanasies prévues dans le cadre de vos nouveaux protocoles?
Answered - 18.05.2022 08:48:56
Thank you for your question. In the amendment VD3221.1c, we have already renounced this method of
euthanasia. We prefer the use of ketamine/xylazine followed by cardiac puncture (which is a method already
described and approved in the parental license).
Qu'entendez-vous précisément par "brève exposition au CO2"?

Answered - 23.09.2021 10:38:29
Thank you for the question. The CO2 procedure was approved in the current version of this license. However, we
acknowledge that the procedure is not recommended anymore unless justified experimentally. We therefore
decide to remove the “brief exposure to CO2” as method used before the cardiac puncture and favor the use of
ketamine/xylazine (which is a method already described and approved in the parental license).

Evaluation of the experiment (Method II) 28-31

28 Recorded parameters
Brief description of the parameter, importance, relation to research question.
Recorded parameters Comments

No Records
29 Experimental set-up and study design
Description of the study design and planning: Number of animals per experiment/series of experiments, number of groups and number
of animals per group. To indicate a description of each group. Specific considerations to include: allocation concealment /
randomization, blinding, sample size calculation (rationale to be entered in section 30), in- and exclusion criteria, definition of primary
outcome variables, statistical analysis plan.
Study A1: total number of animals = 144 mice (12 mice x 3 genotypes x 2 nutritional states x 2
assays)
Study A2: total number of animals = 72 mice (12 mice x 3 genotypes x 2 diets)
Study A3: total number of animals = 24 mice (12 mice x 2 vectors)
Study A4: total number of animals = 120 animals (12 mice x 2 genotypes x 5 developmental
stages)
Study A5: total number of animals = 96 animals (12 mice x 2 genotypes x 4 treatments)
Study A6: total number of animals = 48 animals (12 mice x 2 genotypes x 2 vectors)
Study A7: total number of animals = 48 mice (12 mice x 4 genotypes)
Study B: total number of animals = 60 mice (20 mice x 3 genotypes)
Up to 612 mice (612 GM:) will be necessary to complete the study.
New study A2: The number of animals (72 mice) will not change since we just want to extend the duration
of the experiment.
New study A4: total number of animals = 240 mice (12 mice x 2 genotypes x 5 developmental stages x 2
genders)
Specifically:
Total number of new animals (females) = 120 mice (12 mice x 2 genotypes x 5 developmental stages)
Total number of already approved animals (males) = 120 mice (12 mice x 2 genotypes x 5 developmental
stages)

New study A5: The number of animals (96) will not change, since we would like to replace the taurine
treatment with formate supplementation.
New study A7: total number of animals = 96 mice (12 mice x 4 genotypes x 2 diets)
Specifically:
Total number of new animals (HFHS diet) = 48 mice (12 mice x 4 genotypes)
Total number of already approved animals (CD diet) = 48 mice (12 mice x 4 genotypes )
Up to 780 (612+120+48 =780 GM:) will be necessary to complete the study, we request 168 additional
animals for these changes.
Updated Study A1:
Total number of new animals = 96 mice (12 mice x 2 genotypes x 2 experimental procedures x 2
feeding states)
New Study A1a:
Total number of new animals = 48 mice (12 mice x 2 genotypes x 2 feeding states)
Updated Study A2:
Total number of new animals = 24 mice (12 mice x 2 genotypes x 1 diet)
New Study A3a:
feeding states)
Updated Study A5:
Total number of new animals = 30 mice (30 mothers)
The number of rescued animals (96) will not change, since we are changing the procedure not
adding supplementations metabolites.
However, since we are treating also the mothers, 30 more mice (mothers) will be needed to
ensure enough male pups for the experiment.
Updated Study A6:
Total number of new animals = 48 mice (12 mice x 2 genotypes x 2 constructs)
New Study A8:

feeding states)
Updated Study B:
Total number of new animals = 160 mice (20 mice x 8 genotypes)
New Study B1:
ages x 2 sexes)
Mice of wrong age, gender or genotype:
Total number of new animals = 40 mice
Up to 1834 (780+48+48+12+144+30+48+96+140+320+40+48+12+48+20) will be necessary to

complete the study, thus we request 1054 additional animals for these changes.
New Pilot Study A9:
Total number of new animals = 36 GM mice (12 mice x 3 viruses)
New Study A9:
Total number of new animals = 36 GM mice (12 mice x 3 viruses)
New Study A10:
Total number of new animals = 24 WT mice (12 mice x 2 viruses)
New Study A11:
Total number of new animals = 36 WT mice (12 mice x 3 viruses)

Up to 1906 (1834+36+36 GM) GM + 60 WT, 1966 mice will be necessary to complete the study,
thus we request 132 additional animals for these changes.
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Questions/ Answers
Pourriez-vous préciser l'ordre chronologique selon lequel ces nouvelles expériences seront réalisées et indiquer si les
résultats des unes sont susceptibles d'avoir un impact sur la réalisation des autres ?
30 Rationale for the numbers of animals
Indicate the reasons for the planned numbers of animals, including the method used for statistical analysis(e.g. t-test, ANOVA, mixed-
effects model, etc.). If possible, indicate the test parameters (statistical coefficients, level of significance, power).
It is clear that there is substantial variability between animals in response to various challenges. In
our own experience using previously published work on the C57Bl/6 background, we calculated
with R (a software for statistical computing) using Montecarlo simulation, that in order to achieve a
90% power to detect a difference at p = 0.05, we need at least 12 animals in each group to be
able to perform statistical tests. Only with this number of mice we can approximate to have a
normal distribution to perform statistical analysis such as ANOVA and Student's t-tests. The
isolation of primary hepatocytes is very tricky and does not always yield viable hepatocytes. Often
only one in three isolations are successful. Therefore, in order to be sure that we can get enough
material and considering the possibility of a premature death of the mouse due to this tricky
procedure, we anticipate that we need 20 mice per genotype.

For New Study A4 we plan to use the same number of mice per gender (12 mice/genotype) and the same
statistical power calculation as described above, because we expect to have molecular and metabolic
differences between transgenic male and female mice. New Study A4 will be a pilot study and it will be used
as reference to eventually include more female mice in future studies.
For New Study B1, we plan to use primary hepatocytes from both aged and young mice. We are
asking for 20 mice per genotype per age per experimental procedure using the same rationale
than for study B.
In Study A5, we are asking to include 30 female mice as mothers that will receive water plus
supplements. In our experience, female mice produce around 6 pups. Considering that 50%

female pups and 50% genetic distribution, 1.5 mice will be male Slc25a47hep+/+ and 1.5 will be
male Slc25a47hep-/-. Since we need 12 male mice per genotype, we would need 12/1.5 mothers,
that accounts for 8 mothers. This rationale can be applied for each water supplementation: 2%
choline, 2% betaine and 2% formate. Since we are considering the best-case scenario of 6 pups
per mother and exact mendelian distribution, we would like to ask for 10 mother per condition (3
supplements x 10 mothers) to ensure enough pups for the experiment.
Mice of wrong age, gender or genotype will be used for multiple optimization studies. We would
like to ask for 40 animals, which we suppose will be born of the wrong gender or genotype or will
not be age-matched for the experimental protocols we propose. These mice should be sufficient
for the different studies and trainings we would like to perform until the end of this license. We
cannot be more precise as we do not know yet how long it will take for the optimisation, but we will
report in the AC report what was done with these surplus animals
For Pilot Study A9, Study A9, A10 and A11 we plan to use 12 male mice to obtain the same
statistical power as calculated above. The characterization of the molecular differences between
males and females is not concluded and our studies have been mainly conducted in males.
Therefore, we ask for only males for these studies to complete the validation of the line on mice
where sufficient phenotypic data is available.
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Questions/ Answers
Les études d'optimisation ne devraient-elles pas se faire avant de recruter d'autres animaux dans les autres protocoles de
cette autorisation? Peuvent-elles se faire avec des animaux d'autres lignées?
Answered - 18.05.2022 08:48:56
Thank you for your question. We proposed optimization studies using animals of wrong age, gender or genotype
to eventually reduce the number of mice needed for primary hepatocytes isolation. We do not know if the
idiopathic unsuccessful isolations are indeed due to any procedure method or simply related to animal differences.
We do not have a license where we could study in detail this point and the idea was to use mice that would need
to be euthanized because they do not fit our criteria for experiments for something that may help avoid excess
mice being euthanized in the future. We proposed this method to eventually possibly reduce the number of mice
needed for cellular studies. However, since we cannot know if it could indeed work, and the method we have been
using this far yields viable hepatocytes, we cannot wait for a possible optimization, which may not be possible, to
perform the cellular studies needed to complete this Study. Primary hepatocytes isolation does not in our
experience show particular differences between strains. Hence, optimization can be performed on any strain.
Do you follow the Optimice programme of the EPFL to obtain surplus animals?
Answered - 30.06.2022 12:06:02
Thank you for your question. We do follow the Optimice programme of the EPFL to obtain surplus mice. However,
the Optimice programme offers mainly organs and for our optimizations we need live animals, that will be
anesthetized and immediately euthanised either for primary hepatocytes isolation or freezing methods
optimization. These mice come from surplus mice, mice of wrong age, gender, or genotype. These mice are not
aged matched and would not be used for other assays.

Questions/ Answers
Pourriez-vous faire une description plus détaillée des difficultés que présente l'isolation d'hépatocytes primaires et des motifs
de décès prématuré?Pourquoi choisissez-vous une puissance de 90% plutôt que de 80 ou de 85%?
Answered - 24.04.2020 09:51:49
-The primary hepatocytes isolation is a very delicate technique based on liver perfusion. The mouse needs to be
anesthetized and a catheter is introduced in the portal vein. The liver is then perfused and digested with the
appropriate enzyme solutions (liberase and/or collagenase). Given the tiny size of the rodent's vein, the procedure
is difficult from a technical point of view. In fact, only a few people in our lab have mastered this technique and
even then, it is not rare that the perfusion of the vein goes wrong. If the liver is not well perfused, the quantity of
enzyme reaching the liver will be too low and no primary hepatocytes will be obtained. This is an all-or-nothing
procedure: we obtain either a great number of hepatocytes or none at all. When the perfusion is missed, the
mouse quickly bleeds out and there is no second chance. The experiment has to be restarted all over with a new
mouse.-Having a power of 90% means that if there is truly an effect, there is a probability of 90% to see it. In this
case, our experiments are really costly and time-consuming and we want to be sure that we can detect a true
effect. Using a lower power is riskier and the chance of not detecting an effect (and therefore the chance to repeat
the animal study) is higher. Additionally, we cannot accurately predict the size of the effect that we expect, and
therefore we need a higher power to detect smaller variations.
31 Expertise for statistical analysis
Indicate whether the experimental design and the planned statistical analysis have been verified by a person with expertise in
biostatistics.
No

Handling of animals 32-37

Indicate the effects of the experimental methods on the animals, stress-reducing measures and use of the animals after the end of the
experiment.
32 Downgraded husbandry conditions
Assessment of the necessity of restricted housing (see art. 117 Animal Welfare Ordinance (SR 455.1)): Details and reasons for any
deviations from conditions in which animals are kept as defined in the Animal Welfare Ordinance (SR 455.1).
For the metabolic phenotyping (Study A2 and A7), mice will be single housed to collect feces
during 24 hours. For the measurement of the energy expenditure by indirect calorimetry
(measuring oxygen consumption by the system CLAMS), as well as the measurement of
spontaneous locomotor activity and ingestion of food and water, animals will be single housed for
48 hours (24 hours of habituation followed by 24 hours of measurement) in special metabolic
cages allowing the measurements of all the above-mentioned parameters simultaneously. These
metabolic cages do not have bedding. Mice will be fasted overnight (from 6-7 pm to 8-9 am) for
the GTT experiment. Mice will be fasted for 4-6 hours for the ITT experiment. Given that this
fasting will be done during the light phase, when mice rarely eat, the impact on mouse physiology
should be minimal. Mice will always have freely access to water. The test of cold adaptation will
be performed placing mice in individual cages without access to food or water.
For the short-term cold exposure, mice will be single caged with no bedding and no food but with
water but for no more than 6 hours.
Indicate licence no. of keep-ing laboratory animals: ( If not licenced: housing/husbandry of animals before/during
between/after the individual experiments, structuring, run, feeding and activity, routine inspections by animal
technicians):
Animal facility license: VD H-11.
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Questions/ Answers
Au point 54.1, paragraphe 2. Mesure de la dépense énergétique, vous écrivez "The test can be repeated over several
days". Ici, il n'est question que d'une session de 24 h. Pourriez-vous expliquer?
Answered - 24.04.2020 09:54:31
Yes, probably the phrasing of the procedure is a bit confusing. In principle the CLAMS test could be run for more
than 24 hours, since it is a safe technique and could be used to monitor different parameters. However, in our
specific case, we are going to apply the protocol only for 24h.

Questions/ Answers
Quelles sont les différences entre le test "cold adaptation" et le test "short-term cold exposure" qui
expliquent que les souris ne bénéficient pas des mêmes conditions de détention ?
Pourquoi n'est-t-il dans un cas pas possible d'avoir accès à de la nourriture et à de l'eau ? Et pourquoi dans l'autre cas il n'est
pas possible d'avoir accès à de la litière ?
Answered - 30.06.2022 12:06:02
Thank you for your question, this section was not modified in this amendment. In this license we only perform
short-term cold exposure, the cold adaptation indicated in “Downgraded husbandry conditions” refers to this test
and not the long cold adaptation test, we are sorry for the confusion. Concerning allowing the mice access to food
and bedding, we avoid it since the mice may use these to keep warm and that would invalid the assay. For what
concerns water, there must have been an error in this section, mice are allowed access to water as explained
correctly in section 25.
33 Effects on the animals
Assessment of all expected adverse effects on the animals (art. 19 para. 4 Animal Welfare Act (SR 455)) and of further strain imposed
on the animals through humiliation, through major interference in their appearance or their capabilities or through excessive
instrumentalisation (art. 25 and 26 of the Animal Experimentation Ordinance (SR 455.163)).
We don't expect to influence the well-being of the animals with any of our interventions.
In this amendment, we are requesting mice for AAV8 injection to acutely delete Slc25a47. In this
license, experiments have been performed using Slc25a47hep-/- mice that present a chronic AlbCre
mediated deletion of Slc25a47. Since the Slc25a47hep-/- present good health, we do not expect the
acute models to present a phenotype worse than the Slc25a47hep-/- mice. Still, body weight will be
monitored since Slc25a47hep-/- are leaner, and we expect the AAV8-injected mice to lose a
proportion of their weight due to first the intervention and second the gene deletion.
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Questions/ Answers
N'y a-t-il aucun effet à prévoir pour les animaux sous diète HFHS, quel que soit leur génotype?
Answered - 24.04.2020 09:54:31
The HFHS diet will induce obesity and hepatic steatosis that will be associated with some discomfort for the
animal. For the Slc25a47 KO animals, the effects should be slightly: obesity should be less pronounced since
these animals are leaner on chow diet but the hepatic steatosis should be more pronounced. Please note that this
statement is just an extrapolation of the phenotypes we observed under chow diet.
34 Monitoring of well-being
Assessment of the monitoring and documentation (art. 135 and 144 Animal Welfare Ordinance (SR 455.1)): To indicate are the criteria
for intervention and termination (humane endpoints), and the frequency of checks (who carries out checks, documentation and how
often during which study phase?). Add a score sheet if appropriate.
- by which person(s) Researchers and the caretakers of the different animal facilities (SV, UDP). - frequency
After any intervention procedure, mice will be controlled a few hours later, as well as daily during the two
following days. After that, animals will be checked at least three times a week. Animal caretakers do a visual

check on an everyday basis over the working week and once over the weekend At cage change, a more careful
check is done. Researchers control the mice weekly otherwise.
- criteria of evaluation The weight of the mice will be taken weekly in the long-term experiments.
Any other abnormality or misbehavior will be documented.
For the non-lethal cold test, the body temperature of mice will be monitored every hour for 6
hours. If the body temperature reaches 33 oC, the mouse will be removed from the experiment.
- documentation (e.g. score sheet as stipulated in art. 144 para. 1 TSchV)
- according to the phase of experiment Before the study, animals will be allowed to adapt for at
least one week to the animal house surroundings. If any sign of distress or health problem, the
animal is referred to the CPG veterinarian for further observation, treatment or euthanasia if
conditions get worse. Monitoring might change in case of need at any phase of the experiment.
After AAV8 injection, Slc25a47hep+/+ that receive AAV8_hAAT-Cre+AAV8_hAAT-GFP and

C57BL6/J that are injected with AAV8-shRNA Slc25a47 are expected to become leaner due to
increased energy expenditure. We cannot know if mice delivered with AAV8_hAAT-
Cre+AAV8_hAAT-Slc25a47 and AAV8_hAAT-Cre+AAV8_hAAT-Wars will not display this
phenotype, although we expect AAV8_hAAT-Cre+AAV8_hAAT-Slc25a47 to maintain body
weight. To monitor their weight, mice will be weighted every two days by a researcher and the
scoresheet will be filled. Slc25a47hep-/- are leaner but are otherwise healthy. Hence, we do not
expect these mice to have any distress. Still, if major distress, i.e. score of 3 in the scoring sheet,
is observed mice will be euthanized.
Since the body weight phenotype will be extensively analyzed in Pilot Study A9, to reduce the
stress to the mice given the handling for GTT, ITT and PTT, we propose to weight the miceand fill
the scoresheet once a week in Study A9 unless a higher frequency is needed based on the
observations of the Pilot Study A9.
Lastly, we do not expect effects when Slc25a47-FLAG is expressed in mice by means of injection
of AAV8_hAAT-Slc25a47-FLAG. Still, to ensure mice well-being, mice will be monitored after
injection and body weight will be measured every 2 days at the same time than the scoresheet is
filled.
Document(s): 1
Questions/ Answers
Les données des souris retirées du 'cold test' en raison d'une hypothermie sont-elles utilisables? Selon votre expérience,
quelle est la proportion de souris dont la température baisse jusqu'à 33°C? Ces animaux ont-ils des caractéristiques
communes?Merci d'annexer votre score sheet.

Questions/ Answers
Answered - 24.04.2020 09:54:31
- Yes, they can be included in our results.- About one in every six mice will drop below 33 oC. We could not find
any particular trait linked to this behavior. There does not seem to be any correlations between size, body weight
or age. You will find enclosed the scoresheet for this experiment.
35 Refinement
Assessment concerning implementing rulesunder the specific experimental conditions according to art. 135, 137 para. 4 and 144
Animal Welfare Ordinance (SR 455.1). To indicate the details of mitigating measures or reasons for not using such measures. Which
mitigating measures are taken to reduce stress or minimize anyharm imposed on the animalsunder the specific experimental
conditions?
Any sign of severe illness will exclude a mouse from the experiment, and the mouse will be euthanized (human
endpoints at the score-sheet). In case of potential wounds or infections and other signs of pain, euthanize or
treatment will be discussed with the veterinarian at our institute.
For the short term cold exposure, a drop in body temperature below 33°C (during cold exposure),
as well as occurrence of a general illness.
Mice will be removed from the experiment (sacrificed by CO2 asphyxiation) if they present any of
the following signs: 1) Incapability to move, eat, drink, urinate or defecate without discomfort. 2)
Loss of net weight of 15% compared to a control curve from littermate controls, at the same age.
3) Difficulties to breathe.
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Questions/ Answers
Pourquoi choisir la méthode avec CO2 pour le sacrifice des souris et non une autre méthode ?
Answered - 30.06.2022 12:06:02
Thank you for your question, this paragraph was not changed in this amendment. In the amendment VD3221.1c,
we have already renounced this method of euthanasia. We prefer the use of ketamine/xylazine followed by
cardiac puncture (which is a method already described and approved in the parental license, please see section
27).
36 Distribution by degree of severity
Summary evaluation of the expected maximum stress on the animals (art. 26 Animal Experimentation Ordinance (SR 455.163)):Indicate
the maximum expected degree of severity for each animal category and group. The expected number of animals for each anticipated
degree of severity should be given as a percentage. The allocation is based on FSVO technical information no 1.04.
Severity Level 0 (DG 0): 60 mice - study B Severity Level 1 (DG 1): total 432 mice - studies: A1,
A3, A4, A5, A6 Severity Level 2 (DG 2): A2 and A7= 120 mice Total: 612 mice
For the non-lethal cold test, please note that we are proposing here to increase the body
temperature under which the experiment will be stopped (human endpoint of the experiment). This
is based on our previous observations derived from License VD3187. Indeed, in this license, we
performed several cold test and we can confidently say that when the body temperature of a
mouse reaches 33 oC, that mouse is close to metabolic exhaustion (in our hand, we estimate the
metabolic exhaustion starts when body temperature reaches 32 oC). There is, thus, no need to

wait for the animal to reach 31 oC. We are proposing in this license to increase the human
endpoint of the experiment (33 oC instead of 31 oC) but to keep the experiment as a degree of
severity 2 instead of 3. The rationale will be that in our experiment, no mice will fully reach
metabolic exhaustion. This rationale was accepted for License VD3419.

Updated severity distribution:
Severity Level 1 (DG 1): total 552 (432+120) mice – studies: A1, A3, New A4, NewA5, A6
Severity Level 2 (DG 2): New A2 and New A7= 168 (120+48) mice
Total: 780 mice
Severity Level 0 (DG 0): total 600 (48 + 48 + 48 + 96 + 160 + 160 + 40) - studies: New A1a, New
A8, Update to B, New B1, Mice of wrong age, gender or genotype
Severity Level 1 (DG 1): total 430 (48 + 144 + 30 + 48 + 160) mice – studies: Update to A1, New
A3a, Update to A5, Update to A6, New B1
Severity Level 2 (DG 2): total 24 mice – studies: Update to A2
Total new mice: 1834 mice (660 (60+600) DG0 – 982 (552+430) DG1 – 192 (168+24) DG2)
Severity Level 1 (DG 1): total 132 (36 + 36 + 24 + 36) mice – studies: New Pilot Study A9, New
Study A9, New Study A10, New Study A11
Total new mice: 1966 mice (660 DG0 – 1102 (982+132) DG1 – 192 DG2)
Document(s): 0

Questions/ Answers
Ne serait-il pas possible qu'une souris passe d'une température appropriée à une température lié à un état de
"metabolic exhaustion" en moins d'une heure (les prises de températures était réalisés à ce rythme) ?
Dans ce cas, votre justification du degré de gravité n'est-elle pas invalide ? Certaines souris atteignant effectivement ce
stade grave.
Answered - 30.06.2022 12:06:02
Thank you for your question. In our experience in license VD3187 and VD3419, mice temperature does not drop
of more than 1°C over the course of 1h. Therefore, increasing the point of exhaustion to 33°C impedes the
possibility of animals reaching metabolic exhaustion before the next time point. Therefore, 1h delay between two
measurement is a short enough window to ensure animal welfare.
37 Use of the animals at the end of the experiment
Assessment of the application concerning the implementing rules with regard to the further use of animals after an experiment (see art.
20 Animal Welfare Act (SR 455); art. 141 para 4 Animal Welfare Ordinance (SR 455.1).
Mice will be sacrificed after a brief exposure to CO2 followed by cardiac puncture. For perfusion,
injectable anesthetic (ketamine & xylazine) will be used followed by bleeding.
Mice will be euthanised after anaesthesia using a mix of Ketamine and Xylazine followed by
cardiac puncture for blood collection and organ harvesting.
In Study A5, female mice (mothers) who received normal water will be kept for further breeding,
while mice fed water containing supplements will be euthanized. Female pups will be euthanized
before weaning.
The mice of wrong age, gender or genotype will be euthanized after anaesthesia using a mix of
Ketamine and Xylazine followed by cardiac puncture when freezing tests will be performed. The
mice will instead be euthanized after anaesthesia and bleeding for primary hepatocytes isolation
optimization.
In Study A9, only males Slc25a47lox/lox will be used for experiments, females will be kept for further
breedings.
Document(s): 0

Questions/ Answers
Qu'entendez-vous par "brief exposure to CO2"?

Answered - 24.04.2020 09:54:31
Brief exposure to CO2 is about 2-3 minutes. The time for a mouse to fall asleep and be euthanized.

Rationale and weighing of interests 38-40

Details of the reasons and justifications for selecting the experimental method and animal category.
38 Suitability
Reasons for selecting the animal model with regard to the experimental objective and depiction of scientific validity (i.e. construct
validity, internal validity, and external validity) and reproducibility of the expected findings, if appropriate. Show the extent to which it is
possible to generalise or extrapolate to other study conditions, populations of animals or species, incl. humans.For regulatory
experiments, indicate if an experiment is required by the authorities.
Justification for selection of method or model:
The use of transgenic mice will demonstrate the molecular specificity of the observed effects.
All metabolic testing using non-invasive techniques provide indispensable data for understanding
the function of SLC25A47 in metabolism and disease progression.
Study A2: HFHS diet causes the metabolic challenge that may be needed to see the phenotypic
changes caused by the lack of Slc25a47 in the liver. This diet can be applied in a longer period of
time allowing for the non-invasive phenotyping to be performed. Ultimately, this diet will also
cause lipid accumulation in the liver allowing us to study the impact of the deletion of Slc25a47 in
the development of nonalcoholic fatty liver disease. This used has been used in research for
many years, including in our laboratory. Acute cold exposure in the only way to assess the role of
Slc25a47 in adaptive thermogenesis. This test measures the thermogenic capacities of mice and
the importance of Slc25a47 in this process.
Study A3 and A6: AAV8 vectors use poses as the most convenient way of gene delivery since
they were reported as the most efficient and specific for transduction of the liver without causing
liver damage (increase in ALAT and ASAT).
Justification for selection of animal species:
i) The use of vertebrates in general and mice in particular is justified since only higher multicellular
organisms allow to assess the role of SLC25A47 in terms of hepatic and organismal metabolic
regulation. This statement is based on the fact that this protein is expressed only in vertebrates. In
addition, metabolism is the result of a comprehensive interplay between several organs. ii) The
strong genetic, developmental, physiological and biochemical similarities between mouse and
human, allow us to extrapolate the findings of our study to human physiology and the
development of metabolic-related disorders. This would be more difficult if we would use lower
animal models, such as zebrafish.

Justification for selection of method or model:

New Study A7: HFHS diet causes the metabolic challenge that may be needed to see the phenotypic
changes caused by the lack of Slc25a47 in the liver. This diet can be applied in a longer period of time
allowing for the non-invasive phenotyping to be performed. Ultimately, this diet will also cause lipid
accumulation in the liver allowing us to study the impact of the deletion of Slc25a47 in the development of
nonalcoholic fatty liver disease. This approach has been used in research for many years, including in our
laboratory. Acute cold exposure in the only way to assess the role of Slc25a47 in adaptive thermogenesis.
This test measures the thermogenic capacities of mice and the importance of Slc25a47 in this process.
Justification for selected method or model:
Study A1, A2 and B: Slc25a47 is a gene solely expressed in hepatocytes under physiological
conditions. We have used, so far, a mouse line which presents an hepatocytes specific deletion of
Slc25a47 (Slc25a47hep-/-). When imaging liver samples from these mice using RNA scope, we
found patches of cells which express Slc25a47. Slc25a47hep-/- mice present a deletion
exclusively in the hepatocytes, hence other cell types may express Slc25a47 in pathological
conditions. These cells may affect the phenotype we observe in Slc25a47hep-/-. Hence, in these
studies we would like to include a whole-body knock-out model (Slc25a47CMV-/-). These mice
present a constitutive deletion of Slc25a47; therefore, no cell type can express this gene.
Analysing these mice, we will verify that the phenotype was not affected by cells expressing
Slc25a47.
Study A3a and B: Slc25a47hep-/- mice present a deletion of Slc25a47 in hepatocytes, which is
present since hepatocyte differentiation and albumin gene expression. This model is very useful
and essential for the study of the chronic effects of Slc25a47 deletion. However, the chronic
absence of Slc25a47 affects multiple pathways and it is impossible to understand, using this
model, which ones are directly affected by this gene deletion and which ones are a secondary
effect. To study the very first events and pathways related to Slc25a47 deletion, we would like to
use a more acute approach. Specifically, we would like to use two mouse lines that present an
acute deletion of Slc25a47 in one case in the whole body (inSlc25a47CMV-/-), while in the other it
is hepatocyte specific (indSlc25a47hep-/-). The use of these lines permits to analyse the
hepatocyte specific pathways affected by Slc25a47 absence, while also excluding possible effects
related to other cell types expressing Slc25a47.
Study A8 and B: Slc25a47 deletion in hepatocytes has a detrimental effect on mitochondria, as

observed in Studies conducted under license 3221.1. This result points to a central role of
Slc25a47 in mitochondrial health and function. To study these effects in detail, it would be
important to isolate mitochondria quickly, efficiently, and purely. The efficiency of extraction is

central to maintain mitochondrial health and function. The method we have used thus far is
suboptimal: other organelles can be found together with the isolated mitochondria and enzymes
may be affected by the slower process. To be able to completely understand the role of Slc25a47
in mitochondria, we would like to make use of MITO-Tag mice. These are mice that present an HA
tag on their mitochondria that allows clean and fast isolation of mitochondria. MITO-Tag mice are
crossed with Slc25a47hep-/- mice and relative controls (Slc25a47hep+/+), to generate mice which
present an HA tag on mitochondria specifically in hepatocytes. This model (Slc25a47MITOhep-/-
and Slc25a47MITOhep+/+) is crucial to verify and validate the role of SLC25A47 in mitochondria.
This license is based on Slc25a47lox/lox mice, that have been bred with different mouse lines to
derive different models of interest. Since the paper by Bresciani et al. was published concerns
have been raised about the possible recombination issues that may be present in this line.
Questions have been raised on whether Slc25a47 locus deletion could downregulate its
downstream gene, Wars. However, it would be very difficult to verify the expression of Wars
specifically in hepatocytes. Therefore, we propose two different approaches to understand if the
line is correctly designed. First, we will delete Slc25a47 in Slc25a47lox/lox mice by means of
AAV8_hAAT-Cre recombination. We will then try to rescue the phenotype by using AAV8_hAAT-
Slc25a47 or AAV8_hAAT-Wars. This experiment aims at understanding if the phenotypes we
observe in the Slc25a47hep-/- are solely related to Slc25a47 absence. Still, since we will be over-
expressing both Slc25a47 and Wars, we could also distinguish if any of the phenotypes are driven
by Wars downregulation. Second, we will silence Slc25a47 by means of AAV8-shRNA to avoid
DNA recombination. In details, we will downregulate Slc25a47 mRNA. By using this model, we
can observe if any of the phenotypes are reproduced when DNA recombination is absent. Hence,
we can validate whether no further cofounding factors are present.
We chose to use two models, because, on the one hand, there is a need to understand what the
effects of the Slc25a47 locus deletion are and how they might affect future studies on models
derived from the Slc25a47lox/lox mouse line. On the other, given the purpose of this license, it is
essential to validate which phenotypes are Slc25a47 dependent and which models can be used to
finalize the characterization of this protein’s role in mitochondria and hepatocytes homeostasis
and possibly its function.
Preliminary data also shows that there appears to be a hepatocyte specific maturation of
SLC25A47 in vivo. Since understanding how the protein is processed in vivo may allow us to
further characterize the transported solute and the specific function, we are proposing to use
Slc25a47-Flag tagged constructs to immunoprecipitate the protein to study it in its native form. To
this end, we have designed constructs tagged at the C- and N-terminus of the protein. Once these
are expressed in hepatocytes by means of AAV8_hAAT, we could immunoprecipitate SLC25A47
and further study its structure and function.
Document(s): 0

39 Necessity (3R)
Reasoning why the intended aim of the experiment cannot be achieved by methods that comply better with the 3R criteria. Explain
also why a method that does not require animals does not exist (Replace), why the experiment cannot be carried out with fewer animals
(Reduce), and how all possibilities to reduce the strain on the animals are exploited (Refine).
Evaluation of 3R adequacy of chosen method:
Reduce: After sacrifice of the animals, the blood and all the metabolic organs will be collected and analyzed or
biobanked for collaborations or future projects. The chosen methods allow us to gain a maximum of information
from a minimum of animals (calculated as described in section 54.4). For instance, we will perform RNA
extraction to evaluate gene expression, and protein extraction to assess protein profile. We will also perform
histological, functional assays and phenotyping to characterize the role of SLC25A47 in the context of liver
metabolism.
Refine: As describe in section 53.1, we will use ketamine/xylazine at the indicated doses, to
reduce at maximum the potential pain or distress related to our procedures. In addition, we will
check the mice regularly and provide housing and environmental enrichment (nesting material,
cardboard tunnel and free access to water and food - except for the fasting periods) to reduce
mouse stress.
Replace: Due to the complexity of the biological question, the confined and liver-specific
expression of SLC25A47 and the profound multi-organ interplay involved in the regulation of
metabolic processes, we can't replace the animal experiments described in this license by any
other organism (e.g. yeast, C. Elegans) and by no other procedure (in silico or in vitro). We will
anyway perform in parallel cell culture experiments to investigate the precise molecular
mechanisms at the base of the metabolic phenotype assessed. Additional investigations such as
gene expression, protein profile, histological evaluations, etc, will also be extensively performed
on harvested samples.
Evaluation of experimental method:
Metabolism is a very good example of the complexity of a living organism with collaborating organs. We will use
primary hepatocytes derived from mice as much as possible. The rationale being that with one mouse, ~ 60
millions of primary hepatocytes can be obtained and thus several experiments can be performed. However, we
will not be able to characterize the in vivo function of SLC25A47 in hepatocytes by only using primary cultures.
Therefore, in the proposed research we will use laboratory mice (Mus musculus). The mouse represents an
excellent mammalian model, more similar to the human situation than any other commonly used animal model,
such as zebra fish or fly. Furthermore, this protein and its related pathways are not entirely conserved in lower
organisms. Liver function can be studied in hepatoma cell lines (e.g. HepG2 or Hepa1.6 cells). However, cell
lines are known to differ from the cells in vivo, due to their variations in metabolic genes expression and usage of
carbon sources. More specifically, mitochondrial proteins and important metabolic pathways are changed in cell
lines. The present project aims to study how the regulation of hepatic intermediary metabolism impacts on liver
function. To achieve this, we need living organisms where these pathways are confirmed. Moreover, liver
metabolism is highly affected by the nutritional cues and metabolites released from other organs, making it very
difficult to study in isolated cultured cells.

Additionally, preliminary data demonstrated that the available in vitro models used to study liver
functions present very low levels of Slc25a47 expression, making them an unsatisfactory as a
model for this study.
Document(s): 0
40 Weighing of interests
Assessment of the application with regard to the balance between expected gains in knowledge or other results (interests) and the pain,
suffering,harm, injury or anxiety inflicted (strain on the animals) in accordance with ethical considerations. For more details on the
weighing of interests, see the “Explanatory notes to Form A” and the document “Weighing of interests in animal experimentation” [LINK
www.blv.admin.ch].
By characterizing SLC25A47 and dissecting its role in the hepatic metabolism and organismal homeostasis, we
might unravel a potential therapeutic target for liver diseases, such as non-alcoholic fatty liver disease, which
affects more than 30% of the world population. This is accomplished by the fact that this protein is liver-specific
which brings a new avenue for targeted therapy.
In some of the experiments, mice might experience some discomfort, with the highest severity
grade for an individual procedure being at 2. Other experiments may cause transient pain of low
intensity (such as intra-peritoneal injection). However, we will do our best to keep stress,
discomfort and pain at a minimum level. All parameters that we collect from these experiments will
be extremely informative and useful to prevent and treat the described diseases. Indeed, we are
well aware that our experiments will have some impacts on the health and fitness of the mice and
we expect that the mice fed high fat diet will gain weight and develop obesity and insulin
resistance but the use of animals that resemble the human being is important to have the most
complete picture of how our protein of interest acts as controller of the many metabolic effects
observed in our preliminary results.
Document(s): 0
Questions/ Answers
30% de la population mondiale est affectée par la stéatose hépatique non alccolique? Vraiment?
Answered - 24.04.2020 09:54:56
Yes, indeed, it is true.ReferencesKumar R et al. "Non-alcoholic Fatty Liver Disease: Growing Burden,
Adverse Outcomes and Associations." J Clin Transl Hepatol. 2020Loomba R&Sanyal AJ. "The
global NAFLD epidemic". Nature Reviews Gastr.&Hepat. 2013Younossi Z et al. "Global burden of
NAFLD and NASH: trends, predictions, risk factors and prevention". Nature Reviews Gastr.&Hepat.
2017

List of documents
Type Title Description Filename Date
Animals GMA forms 3221.1 Datasheets initially GMA forms 3221.1 17.09.2021 07:51:08
combined.pdf submitted in answers combined(1).pdf
but mandatory here for
the SC
Animals FormD_184_V1 FormD_184_V1.pdf 02.05.2022 15:10:44
Answer GMA forms 3221.1 GMA forms 3221.1 GMA forms 3221.1
combined.pdf combined.pdf combined.pdf
Answer New study A7 New study A7 23.09.2021 10:28:35
modified.pptx modified.pptx
Answer Timeline.pptx Timeline.pptx 16.05.2022 17:02:19
Answer REVISE_1.pdf REVISE_1.pdf 30.06.2022 11:27:53
Answer Score sheet Cold Score sheet Cold Score sheet Cold
exposure.pdf exposure.pdf exposure.pdf
Application Answers to Answers to 23.09.2021 10:30:24
questions_3221.1c.do questions_v4.docx
cx
Application 3221.1 3221.1 16.09.2021 23:55:10
amendment_15.09.21. amendment_15.09.21
docx _v4.docx
Application New study A7 New study A7 23.09.2021 10:29:15
modified.pptx modified(1).pptx
Application New workflows-3221 New Work flows.pdf 01.04.2020 16:51:45
Application Animal Sheets_GMA Animal Sheets_GMA Animal Sheets_GMA 02.03.2017 08:39:49
forms 01032017 forms 01032017 forms 01032017.pdf
Field New Work flows_new New Work 16.09.2021 23:38:34
Figures_amendment.p flows_amendment -
df New
Figures_amendment.p
df
Field New New 02.05.2022 08:56:34
Workflows_V3_LAST. Workflows_V3_LAST.
pptx pptx
Field Score Sheet Score Sheet 06.01.2023 16:16:13
Study_weekly Study_weekly
weighting_v2.docx weighting_v2.docx
Field 3321.1e_v5.pptx Workflow for 3321.1e_v5.pptx 09.01.2023 08:50:32
Amendment January
2023

2023 01 12 - 07 47 42 - FormA - VD3221x1e

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Form A National no. Cantonal no.

Application for licence to perform animal experiment 32387 VD3221x1e

Name EPFL SV IBI

Name Julie Parchet-Piccand

Name Kristina Schoonjans

02 Address of the cantonal authority

Will the experiment be performed in No

If yes: Secondary canton(s) none

Exported from animex-ch at: 12.01.2023 07:47:41 1 / 57

Application for animal experiment 32387 VD3221x1e

Indication of application title Study of the role of Slc25a47 in hepatic metabolism

05 Title for the publication

Corresponding application type Supplementary application

07 Maximum prospective degree of severity

Indication of the maximum 2

Please find attached the updated GMA forms.

08 Duration of project and date of start

Duration of project 3 years / 0 months / 0 days

Exported from animex-ch at: 12.01.2023 07:47:41 2 / 57

Application for animal experiment 32387 VD3221x1e

Data sheet info and related forms

Origin of the animals

Place where the animals are kept

Data sheet info and related forms

Origin of the animals

Place where the animals are kept

Exported from animex-ch at: 12.01.2023 07:47:41 3 / 57

Application for animal experiment 32387 VD3221x1e

10 Location of the experiments

Address Room number Description

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Application for animal experiment 32387 VD3221x1e

Name Role Qualification Deputy Area of responsibility

Name Julie Parchet-Piccand

13 Principal Study Director

Name Kristina Schoonjans

Exported from animex-ch at: 12.01.2023 07:47:41 5 / 57

Application for animal experiment 32387 VD3221x1e

Name of Deputy Study Director Johan Auwerx

14 Animal Welfare Officer of Institute

Name Julie Parchet-Piccand

Exported from animex-ch at: 12.01.2023 07:47:41 6 / 57

Application for animal experiment 32387 VD3221x1e

Purpose of the experiment 15-22

Indication of the scientific field Physiology

17 Association with diseases or disorders

The project is associated with Human diseases:

18 Associated procedures required by law

Indication of the regulatory No association with procedures required by law

Has the project been appraised? Yes

20 Objective of the experiment and background

Exported from animex-ch at: 12.01.2023 07:47:41 7 / 57

Application for animal experiment 32387 VD3221x1e

Exported from animex-ch at: 12.01.2023 07:47:41 8 / 57

Application for animal experiment 32387 VD3221x1e

C. Koliaki et al., "Adaptation of Hepatic Mitochondrial Function in Humans with Non-Alcoholic

F. Palmieri, "The mitochondrial transporter family (SLC25): physiological and pathological

M. G. K. Tan, L. L. P. J. Ooi, S. E. Aw, and K. M. Hui, "Cloning and identification of hepatocellular

Exported from animex-ch at: 12.01.2023 07:47:41 9 / 57

Application for animal experiment 32387 VD3221x1e

21 Results of the previous application

SC-32387-VD3221x1d - Last submitted to canton: 30.06.2022