Problem Set 5: Mice
Due: Monday, November 28, 5pm (NO LATE PROBLEM SETS ACCEPTED!!!)
 Turn in problem sets to the Agard lab, room S414, in the box on Elena’s desk
 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.
Motor neurons in the spinal cord innervate voluntary/skeletal muscles to control movement.
Your work suggests that developmental patterning specifies motor neurons to innervate specific
muscle groups only. To identify the genes that might control this process you’ve done RNAseq
on the motor neurons that innervate the mouse biceps. The RNAseq shows a novel proteincoding transcript encoded by a gene containing a single coding exon; you name this locus
popeye. Popeye message is only expressed in the spinal cord in motor neurons that innervate the
biceps, something you determine with in situ hybridization studies.
You wish to genetically label popeye expressing neurons with a fluorescent reporter (say
tdTomato) so that ultimately you can do electrical recordings from them while the mouse is
moving about.
1) Design a knock-in gene conserving strategy to label the biceps motor neurons. For this
answer you only need to draw the construct – but please be specific with construct details.
Your 129 ES cells target well, the injection goes smoothly, and you get great male chimeras.
These yield lots of F1 brown progeny upon mating to standard WT B6 mice (black coat color).
Strangely, you find via PCR that of the 15 male and 16 female progeny (across 4 litters), only the
females carry the targeted allele.
2a) What do you think is going on?
2b) If your hypothesis is correct, what fraction of F1 brown female progeny should carry
the targeted popeye allele?
2c) What fraction of the F1 male progeny of these females (crossed to WT males) will carry
this allele?
Once you’ve crossed these brown females to WT males you analyze resulting progeny bearing
the targeted popeye allele for expression. In complete contrast to your RNAseq and in situ
studies, you now find tdTomato expressed in all motor neurons and also in many other cell types
in other tissues.
3) Why this aberrant expression and how do you take care of it?
You’ve now fixed the aberrant expression problem and go back to examine tdTomato expression
in vivo. To your relief only the biceps motor neurons express tdTomato in the spinal cord.
You’re super careful (or just plain damn lucky) and find out to your surprise that in fact even the
biceps muscle expresses tdTomato.
4) How do you test whether or not this is aberrant tdTomato (and therefore popeye)
expression in the biceps muscle?
You’re happy that your experiments show that the tdTomato expression in the biceps is not
misexpression. You’re now intrigued by what the function of popeye might be and you put aside
your dream of recording from the tdTomato expressing motor neurons. You design a knockout
construct for popeye, target ES cells, and generate chimeras. You get 3 excellent, really brown
male chimeras that have difficulty walking about because of weak forelimbs. Fortunately they
mate and go germline and you get lots of brown male and female progeny, all of whom walk
about normally.
You now analyze the male chimeras and find that the biceps muscle is fine but the innervating
motor neurons are all missing. The same is true for homozygous null females. Curiously in
popeye/- females, the number of the motor neurons is reduced to 50% normal, and half of the
surviving motor neurons express popeye and the other half do not.
5a) Provide an explanation for your findings (hint: Where does popeye function to control
motor neuron survival?)
5b) Design a genetic strategy to test your hypothesis. Be specific with construct details,
whether you will be using homologous recombination or random integration events, and
detail the breeding strategy and the control animals you will use. (Hint: you will need to
generate two sets of mice. Assume you have access to appropriate promoters.)