Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Standard view
Full view
of .
Look up keyword
Like this
1Activity
0 of .
Results for:
No results containing your search query
P. 1
Gene Dosage Sensitivity is revealed through analysis of a hypomorphic Hand1 allele

Gene Dosage Sensitivity is revealed through analysis of a hypomorphic Hand1 allele

Ratings: (0)|Views: 5|Likes:
An essay for the 2011 Undergraduate Awards Competition by David McConville. Originally submitted for Placement: Diploma in Industrial Studies BMS504 at University of Ulster, with lecturer Dr. Clifford Stevenson in the category of Medical Sciences
An essay for the 2011 Undergraduate Awards Competition by David McConville. Originally submitted for Placement: Diploma in Industrial Studies BMS504 at University of Ulster, with lecturer Dr. Clifford Stevenson in the category of Medical Sciences

More info:

Published by: Undergraduate Awards on Aug 29, 2012
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
See more
See less

02/24/2014

 
 
Page 1
 
Gene dosage sensitivity is revealed through analysis of ahypomorphic
 Hand1
allele
 Hand1
gene dosage is crucial for embryonic viabilityKey Words: Hand1, extraembryonic mesoderm, hypomorphic, heart, Hand2.
A hypomorphic
 Hand1
allele was unintentionally generated when an attempt wasmade at creating a
 Hand1
cDNA knock-in reporter mouse. This hypomorphicallele as a homozygote causes an extention of the embryos survival to aroundE12.5. Heart development appears typical the exception being a thinner leftventricular myocardium. The allantois appears larger than in wildtypes. Thesephenotypes are in-line with current thinking regarding systemic
 Hand1
geneablation. Analysis of 
 Hand1
mRNA shows expression levels at around 30% thatof wildtype littermates. The data indicated a threshold of 
 Hand1
expressionnecessary for embryonic viability.
 Hand 
factors are essential for the control of embryonic development, pathologicaldisease and tissue specificity.The
 Hand1
gene encodes a basic helix-loop-helix (bHLH) transcription factor withexpression being perceived to be restricted to placental trophoblast cells pre-embryonic day 7.5 (E7.5).
(1)
The bHLH protein by design allows for dimerization-both as homodimers andheterodimers with other proteins within the family. (2)
 
Studies have noted that
 Hand1
is prevalent in distal parts of the lateral mesoderm postE7.5 and is detectable in the developing heart tube and pericardium.
 Hand1
’s
importance in embryogenesis is shown through its essential contribution to heart andSNS development; as well as having a role in extraembryonic mesoderm formation.
(3)
 A
 Hand1
knock out embryo (2 null alleles/ homologous null) is lethal at mid-gestation; usually E9.5. This has been found to be due to complications with the extra-embryonic mesoderm and the cessation of cardiac development at the heart-loopingstage. Rightward looping of the heart gives rise to the presumptive left ventricle. Dueto
 Hand1
expression in myocardial curvature it can be stated that Hand1 plays asignificant role in left ventricle formation.Post E11.5
 Hand1
is expressed in the lateral mesoderm, distal portions of the limbbud and in the neural crest cells.
(4)
These migrate out of the closing neural tube andcontribute to structural parts of the face and smooth muscle of the pulmonary artery.
(5)
 Further validation of the role of 
 Hand1
has been conducted using over-expressionmodels. By over-expressing
 Hand1
in
 Hand1
expressing cells the outer curvature of the ventricle is enlarged thus giving irregular looping and ventricular deformities.This is most likely to be due to greater myocyte density.
(6)
 By using
 Hand1
conditional knock-outs with
Wnt1-Cre
to remove the functional
 Hand1
allele in all neural crest cells, previous studies have shown that this does not
 
 
Page 2
 
affect facial or cardiac phenotypes concluding that
 Hand1
expression in neural crestcells is not essential for normal development.However, by placing this conditional knockout on a systemic null
 Hand2
backgroundthe embryos do in fact have both facial and exacerbated cardiac phenotypes. Thispoints to the idea that a gene dosage relationship is important in neural crestdifferentiation.
(7)
 Placental Lactogen1 (
PL1
) is a critical growth factor with regard to both embryonicand extra-embryonic tissue. In
 Hand1
null embryos
PL1
is reduced causing vascularformation within the yolk sac to cease soon after it begins.The initial idea was to create a
 Hand1
marker allele that would not behaploinsufficient for
 Hand1
. In order to show Hand1-expressing cells, a cDNA-knock-in approach with an IRES eGFP cassette was undertaken. The
 Hand1
 homozygous cDNA knock-in mice are not viable and the lethality is not rescued bythe removal of the neomycin cassette.It was however found that these homozygous knock-in embryos exhibit a definiteextention of survival to between E10.5 and E12.5; different from
 Hand1
systemicknock-outs. Phenotypic outcomes include irregular development of the caudalregions, thinner and more dilated hearts. Furthermore, due to
 Hand1
’s role in extra
-embryonic tissue the tissue and yolk sacs displayed phenotypes. Using mRNAanalysis it was found that
 Hand1
cDNA knock-in alleles are hypomorphic with
 Hand1
expression being between 30% and 40% that of wildtype littermates. This isindicative of a threshold of necessary
 Hand1
expression. As was expected,
 Hand2
 was upregulated when
 Hand1
expression decreased. This creates an imbalance withregard to bHLH stiochiometry.
Materials & Methods
Plasmid Constructs
The
 Hand Hand1
+neo
targeting vector employed the
identical 5’ and 3’ targeting arms
employed in generation of the
 Hand1
 LacZ 
 
knock-in (5). Inserted between these arms isa
 Hand1
cDNA that
 beginning 70 base pairs 5’ of the
initiating methionine andending at the translational stop site. The
 Hand1
cDNA is immediately 5’ of an
 IRESGFP cassette derived from pIRESeGFP
(Clonetech), which is immediately 5’ of 
a PGKNeo cassette flanked by loxP sites. Homologous recombination was achieved ata frequency of one in fifteen and two independent ES lines were injected andproduced germline transmission with indistinguishable phenotypes.
 Mouse strains Hand1Hand1
+neo
 
mice were generated by the IU knockout-transgenic core from EScells targeted with the construct described above.
 Hand1
 Hand1
Δ
neo
mice weresubsequently generated via intercross of 
 Hand1
 Hand1+neo
 
mice with mice heterozygousfor
 
 
Page 3
 
 EIIA-Cre
allele (9). Both versions of the reporter allele are viable and fertile asheterozygotes.
 Hand1
 LacZ 
 
knock-in mice have been reported previously (5) and
 Hand2
knockout (10) mice have been reported previously.
 Histology
Harvested embryos ranging from E9.5 to E11.5 were initially fixed in 4%PFA thenprogressed through an ascending alcohol gradient and mounted in paraffin wax. Thesewere then sectioned at either 7µm or 10µm and stained using Mayers Haematoxylinand Eosin Y. Slides were ran through Citrisolv
©
to remove the wax, descendingalchohol gradient and distilled water to rehydrate. Haematoxylin staining wasperformed followed by bluing of sections using running tap water. Counterstainingusing Eosin Y was carried out prior to dehydration through graded alcohol beforebeing coverslipped with Permount
©
.
 In-situ Hybridisation
 – 
qRT PCR
Using 10µm paraffin sections, Digoxygenin(DIG) labelled section in-situhybridisations were performed with T3, T7 or SP6 polymerases and DIG-labellingmix. ISH uses a labelled complimentary RNA strand to localize a specific DNA/RNAsequence in a portion of tissue. Sample cells were treated to fix the target transcriptsin place, thereby increasing probe access. The riboprobe hybridises to the targetsequence at a temperature of 70
o
C. Digoxygenin(antigen labelled base) the labelledprobe is localised and quantitated in the tissue. The basic stages involve binding of 
mRNA’s to the RNA probe followed by binding antibody
-phosphatase to the RNAprobe. The antibody is then stained. Sense and anti-sense DIG-labelled riboprobeswere transcribed for Hand1, Hand2, eGFP and PL1. qRT-PCR was done on aLightcycler 48011 using Taqman labelled proprietary primer sets for Hand1, Hand2and Gapdh as internal control. Embryos, yolk sacs or placenta were flash frozen andgenotyped via genomic DNA from the yolk sac or head of the embryo. RNA wasisolated using a HP RNA tissue kit from Roche ad cDNA was prepared usingtranscriptor first strand cDNA synthesis kit.
TUNEL & Immunohistochemistry
TUNEL is a method used to detect DNA fragmentation as a result of apoptosis, bylabelling the terminal ends of nucleic acids. Analysis was performed on sectionedembryos ApopTag Plus Fluorscein in situ apoptosis detection kit. We also assayedcell proliferation using Bromodeoxy Uridine (BrdU) detection. 5-bromo-2-deoxyuridine is a synthetic nucleoside and also an analogue of thymidine. The mostcommon use for BrdU is in the detection of proliferating cells in living tissue. It canbe incorporated into newly synthesised DNA of replicating cells substituting thethymidine during the replication process. Antibodies specific for BrdU are used todetect the incorporated chemical. This defines cells that are actively replicating theirDNA. What is of note is that the cells must first be exposed to acid or heat in order todenature the DNA and allow binding of the antibody.

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->