BME 430: Tissue Engineering

Animal Models
Fall 2022
 What different types of
animal models are there?

2
History of the Mouse Model
• Lucien Cuénot - Mendelian inheritance coat colors in mice
(1902)
• William Castle - coat color genetics in mice (1903)

• Castle’s student, Clarence Little, is credited with conceiving of

and creating the first inbred strain of laboratory mice (DBA,
named for its coat color genes: dilute, brown, nonagouti) to
unravel the genetics of cancer

• Little also created the C57/C58 family of strains and started

Jackson Laboratory in 1929

3
 Mouse Models: A Tale of Mice and Men
• Roles: concept development,
validation of mechanism of
action, safety/efficacy
• Ultimate driver – unique
sequence variations dictate or
predict diseases

• Genetic and physiological

similarities
• Of ~30,000 genes only 300
are unique to either species

4
Doyle A, McGarry MP, Lee NA, Lee JJ. Transgenic Res, 2012
Advantages
• All mice are generally on the
same genetic background
(minimizing background noise)
– many strains are inbred
• Inter-generational interval is 3
months
• Identical environmental
exposures (housing, treatment,
atmosphere/cage)
• Identical experimental
protocols, endpoints, evaluation
of physiological parameters

5
 Disadvantages
• In general, diseases display “on again-off again” gene
expression
• … Strategies for doing this – Tetracycline/Docycycline inducible mice
• Mice live ~2 years, humans live ~80 years
• Relevance to humans is not always perfect, examples:
• Mouse brain: 70 percent neurons and 30 percent glia, while the
human brain has the opposite ratio
• Mouse brain’s surface is smooth, while the human brain has wrinkles,
ridges, and crevices, a design that may allow us to pack more neurons
into the skull’s restricted space.

https://www.hopkinsmedicine.org/institute_basic_biomedical_sciences/news_events/articles_and_stories/ 6
model_organisms/201010_mouse_model.html
Genetic Models: Duchene Muscular Dystrophy (DMD)
• Sex-linked disease that affects boys
• Increased muscle damage and weakness

• Mouse model: mdx-/-

• Produces nonfunctional dystrophin
• Replicates phenotype of disease
• However:
• Does not replicate reduction in life
expectancy (25% mdx-/- vs. 75% human) McGreevy et al. Dis Model Mech, 2015

• Does not replicate pain

• Newer models addressing these points
7
Genetic vs. Surgical Induction of Phenotype
• Spontaneous Hypertension Models
• Useful to study cardiovascular system
• Cardiac hypertrophy/failure
• Renal dysfunction
• Increased risk of stroke

• Genetic inbreeding of hypertensive animals Transverse aortic

construction (TAC)

• Select for phenotype

• Surgically constrict vessels, intentionally
• Generate phenotype

• Reliability of models (and off-target effects)

8
Diet Induced Models: Obesity and Diabetes
• High fat content (protein constant)
• At least 1 week required to adopt
phenotype

• Useful for metabolic studies

• Pre-diabetic (type 2)
• Impaired glucose tolerance
• Obesity and hypertension

• Usefulness of rapid phenotype

change?
• Introduction of genetic factors
9
 Mimicking Long Term Diseases: Alzheimer’s
• Relatively unknown etiology
• Take years to develop symptoms/disease
• Familial vs. sporadic cases

• First model of Alzheimer’s over-expressed

V717F β-amyloid precursor protein
(sporadic onset)
• New animal models include Tg2576
(familial onset)

• Difficulty in replicating diseases that we

know little about (and impact of TIME)
10
Games and Adams et al. Nature, 1995
 The Humanized Mouse
• A mouse that has either:
• Several human cells
• Short strands of human DNA
• Human tissues (or tumor)
• Humanized immune system
• or parts of the human microbiome
• Require: highly immunodeficient
mice
• Nude
• Severe combined immunodeficiency
(SCID) mice
• Immune reaction is not the same
as in humans
• More recently - introducing
human cytokine genes
(NOD/SCID/γcnull and
Rag2nullγcnull mice) to improve the
accuracy of the immune response

11
Ito R, Takahashi T, Katano I, Ito M. Cell Mol Immunol, 2012
 Single Nucleotide Polymorphism (SNP)
• Is a single base which differs between two genomes, and the
DNA around that base is otherwise unchanged

Codon - a sequence of
three nucleotides which
together form a unit of
genetic code in a DNA
or RNA molecule

= Synonymous
12
http://thegenomefactory.blogspot.com/2013/10/understanding-snps-and-indels-in.html
 INDEL (INsertion/DELetion)
• Where a single base has been deleted, or inserted, into one
genome relative to another
• Symmetrical relationship, as a deletion in one corresponds to an
insertion in another

13
http://thegenomefactory.blogspot.com/2013/10/understanding-snps-and-indels-in.html
 INDEL (INsertion/DELetion)
• Result in frameshift mutations
• Usually either extends …

Stop codon

14
http://thegenomefactory.blogspot.com/2013/10/understanding-snps-and-indels-in.html
 INDEL (INsertion/DELetion)
• Result in frameshift mutations
• Usually either extends or truncates

15
http://thegenomefactory.blogspot.com/2013/10/understanding-snps-and-indels-in.html
Concept Check! SNP or INDEL?

• Sickle Cell Anemia

Normal:
Nucleotide: CAG GCC CTT GAA AGG AAT…
Amino Acid: Q A L E R N
• Crohn’s Disease Crohn’s Disease:
Nucleotide: CAG GCC CCT TGA AAG GAA …
Amino Acid: Q A P -

16
Methods of Inducing Mutations
• Spontaneous (infrequent)
• “Forward” genetic approaches – initiation of a mutagenic event
followed by screens to identify mice with altered genetic traits
• Early approaches: radiation, chemical treatments
• Now: directed gene recombination events
• “Reverse” genetic approach – a gene of interest is identified
from it’s association with human disease and then becomes a
target in mice
• Transgenic mice, Knock-out/Knock-in mice

17
Reverse Genetic Approach
• Transgenic mice – introduce
exogenous genes or DNA
sequences (trans gene) which
integrate as a single
chromosomal insertion event
that becomes heritable

• Mendelian Trait
• “Basic” hereditary principles
• Punnett squares

18
Reverse Genetic Approach
Mice generated by the genetic manipulation of ESCs such that a
specific genetic locus is targeted and …

• Knock-out … rendered non-functional either by the insertion of

irrelevant DNA sequence information to disrupt the expression
of the encoding locus or by the deletion of DNA sequence
information from the targeted locus

• Knock-in … has been altered either by the one-for-one

substitution of DNA sequence information or by the addition of
sequence information not found in the endogenous genetic
locus

19
 The Creation of Transgenic Mice

20
Doyle A, McGarry MP, Lee NA, Lee JJ. Transgenic Res, 2012
 The Creation of Knock-out Mice

Founder: Animals (and

offspring) whose genome
has been altered by
genetic engineering

21
Doyle A, McGarry MP, Lee NA, Lee JJ. Transgenic Res, 2012
Cre-Lox – Most Common Reporter System
• Tissue-specific expression
• Inducible knockouts/knockins

• It can:
• Turn transgene expression on/off Retinal ganglion cells in a mouse
• Track individual cells/lineages (Joshua R. Sanes)

• Inversions
• Translocations
• Report gene expression

cerebellum gyrus (Tamily A. Weissman)

https://augenblickblog.com/2017/02/20/over-the-brainbow/
22
 Cre-Lox for Knockout
• The Cre/lox system can be used to turn off a transgene

• Such a transgene is designed with loxP sites flanking all or part

of it (“floxed” transgene)

Cre recombinase
removes gene

23
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
 Example Schema for Tissue Specific Knockout
1st step: generate mice that are heterozygous for a loxP-flanked
allele and hemizygous/heterozygous for the cre transgene

Hemizygous: Having only a

single copy of a gene instead
of the customary two copies

~50% of the offspring:

• Heterozygous for
the loxP allele
• Hemizygous/heterozygous
for the cre transgene

24
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
 Example Schema for Tissue Specific Knockout
2nd step: generate mice that are homozygous for a loxP-flanked
allele and hemizygous/ heterozygous for the cre transgene

~25% of the offspring:

• Homozygous for the loxP-
flanked allele
• Hemizygous/heterozygous
for the cre transgene

25
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
Checkpoint: Which is the Desired Experimental Animal

1.

2.

26
 Cre-Lox for Knockin
• Insertion of a loxP-flanked “stop” sequence (transcriptional
termination element) between the promoter and transgene
coding sequence blocks the expression of the gene
• Cre recombinase removes the stop sequence, so the transgene
is expressed only in the cells where Cre is active

27
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
 Cre-Lox Knockin Example: Breast Cancer
Cre recombinase in
mammary tissue

Kras expression in
mammary tissue

28
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
 Examples of Tissue-Specific Cre-Lox Mice

29
Lewandoski M. Nat Rev Genet, 2001
 Cre-Lox Reporter Strains
Myh6: cardiac alpha
(α)-myosin heavy chain

Reporter strains useful for:

• Lineage tracing
• Tracking transplant tissue
Tissues in targeted • Monitoring cell activity
area express GFP • Tracking “regeneration”

30
https://www.jax.org/news-and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies
Time Dependent Considerations of Expression
• Comparative study of gene function (on- vs. offset)
• Mimic a pathology with late onset
• Studying embryonic lethal conditions

Does the inducer have side effects?

31
Conditional Expression of Genes: Tet Systems
• Tet-On system: the transgene is turned on (induced) by
administering tetracycline (or it’s derivative doxycycline) via
drinking water and turned off (suppressed) by withdrawing
from the drinking water
• Tet-Off system: tetracycline/doxycycline prevents the tTA
transcription factor from binding DNA at the promoter
inhibiting gene expression

https://www.genoway.com/technologies/tet/tet-system.htm 32
Tet-On Mouse
TRE = Tet Response Element

https://www.takarabio.com/learning-centers/gene-function/inducible-systems/tet-inducible-systems/tet-systems-overview
33
Lessons from Mice
• Replicating genetic deficiencies that cause human diseases

• Knockout mice – genes involved in disease are also involved in

development (ex: Brca-1)

• Genetic functions are redundant – loss of function had little

effect on mice

• Loss of function can be lethal if expressed in all the tissues (not

just the tissue of interest)

34
 Beyond the Mouse…
• Rat is next most common model: larger than mouse, preferentially
used in neurobiology, cardiology, immunology and toxicology
• Larger mammals (rabbits, cats, dogs, pig, sheep, monkeys) used
depending…
• Size important for anatomy, surgical
procedures, analysis
• Balance between cost and biologic relevancy

Clinically relevant organ size(s)

35
Gilbert-Honick et al. Adv Healthcare Mater, 2020
 TOMORROW IS
ELECTION DAY

VOTE – LET YOUR

VOICE BE HEARD