You are interested in the development and function of the spleen. Your reading of the literature
suggests that the development of many, if not most, major cell lineages and organ types is
controlled by a basic helix-loop-helix (bHLH) type transcription factor. The mouse genome only
encodes 55 bHLH transcription factors. You do RT-qPCR for each of these 55 genes, using
mRNA from tissue crudely dissected from the site of spleen development. To your delight your
qPCR detects message for only 1 bHLH encoding gene, which you name asplenia.
You then do in situ hybridization for asplenia to examine expression in the embryo at the site of
spleen development.
1) Why bother doing in situs if your qPCR already shows expression?
Need to confirm that it is indeed expressed in the precursor tissue to the spleen. Your crude
tissue dissection to generate mRNA for the qPCR could easily have picked chunks of other tissue
that may not give rise to the spleen.
Your in situ shows strong expression in the precursor tissue to the spleen at E13.5 (embyronic
day 13.5), confirming your qPCR results. You now want to test the function of asplenia in vivo
and also use it as a tool to study spleen development and physiology. To this end, you obtain the
genomic DNA fragment encompassing the asplenia locus and figure out that there is a single 5’
non-coding exon and a single coding exon. You design a lof allele, replacing exon 2 with an inframe EGFP. And you design a tagged allele, inserting an IRES-EGFP 3’ of the stop codon in
exon 2, in order to obtain highly enriched populations of spleen cells for cell biological analysis
of spleen function in the adult animal. This is a cool experiment because the spleen contains a
lot of blood and absent a specific marker for spleen cells, it has been previously impossible to
specifically isolate spleen cells and study them.
The chimeras carrying your lof allele do not transmit the allele through the germline, although
they seem to mate OK with the females. Examination of the testes of these chimeras reveals no
viable sperm. By contrast, chimeras bearing the tagged version provide germline transmission
just fine.
2a) Provide 2 genetic explanations of the failuer to transmit the lof allele.
- Haploinsufficiency phenotype in sperm viability if asplenia is autosomal
- asplenia is an X linked gene required for sperm viability.
2b) The litters generated by your chimera carrying the tagged allele (asplenia-IRES-EGFP)
provide an easy way to determine which of your 2 genetic explanations above is the correct
one. How?
- If autosomal, then chimera should transmit the tagged allele to males and females, 1:1.
- If X linked, chimera should transmit the tagged asplenia only to females. (Reasoning being
that standard ES cells are male.)
3) Redesign your genetic strategy for the lof allele to bypass the sperm viability problem so
that you can get down to studying the role of asplenia in vivo. Assume you have access to
all/any promoters you might conceive of using.
3a) Please provide the two constructs you will have to make to do your studies. Indicate
whether the resulting mice will be transgenic or generated by gene targeting.
Gene targeting: 5’arm-exon1-loxP-exon2-loxP-neomycin selection-3’arm
Transgenic: Spleen promoter-Cre recombinase-IRES-lacZ-pA (SpCre allele)
TAs, the IRES-lacZ is optional, but any reasonable person would likely incorporate lacZ or even
better a fluorescent reporter (mCherry, tdTomato) to mark expression of Cre.
3b) What are the control animals for experimental analysis of mice with a deletion of
asplenia that have been generated with your strategy? Please provide explanations for why
these are the relevant controls.
- WT, for obvious reasons
- Aspleniaflox/flox, to test for partial lof etc (because genetic modification causes misexpression
etc) in absence of Cre
- Your transgenic control alone (SpCre ). In many cases the transgenic insertion can land in a
locus or a regulatory region and disrupt normal development, differentiation, function etc.
3c) Please provide specific details of the mouse breeding you will have to do to generate a
deletion of asplenia as well as the control siblings for experimental analysis. What is the
expected frequency of the experimental and control siblings from the cross?
Aspleniaflox/+ x Aspleniaflox/+, SpCre/+.