Problem Set 5: Mice
Due: Monday Dec. 3, before 5 PM to Weiner Lab
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Please be explicit and detailed in your answers in order to receive full credit.
Please type out answers (you can draw in any diagrams).
Have your name, question #, and page number (example: John Smith, Question #1, Page
1 of 2) on each page.
Start each question on a new page and staple each question together separately.
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?
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 failure to transmit the lof allele.
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?
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.
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.
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?