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Unformatted text preview: Zoo 470 2008 Problem Set #3
Name:________________________________ Student Number:__________________ Page 2 If you worked in a group, other collaborators: _____________________________________________ 1. Nanog encodes a homeodomain-containing protein, and is thought to be necessary for maintenance of pluripotency only after cells in the mouse embryo become committed to either trophoblast or embryonic fates, and is not expressed until this time. a. What technique could you use to detect where Nanog mRNA is expressed in the early embryo? (1 point) Technique: in situ hybridization b. In what cells of the "morula" stage embryo (i.e., the 16-32 cell embryo) would you expect Nanog mRNA to be expressed in? State your reasoning. (3 points) Interior cells, which are pluripotent, and give rise to the ICM of the blastocyst c. What cells in the later mouse embryo (i.e. after organogenesis is underway) would you expect to express Nanog mRNA? State your reasoning. (2 points) Primordial germ cells, which retain pluripotency. [partial credit for epiblast, because the problem asks about after organogenesis] d. Where within the cells that produce Nanog protein would you expect it to be found? (1 point) Location within cells: Nucleus (it's a transcription factor) 2. You set out to examine the relationship between Nanog and Oct-4. Design an experiment that would test whether Nanog expression requires Oct-4 function. Clearly state your experimental procedures, and your expectations if Nanog expression depends on Oct-4 function. Assume you can perform any experiment involving mouse cells discussed in class, and that you can detect gene expression using any technique discussed in class. (5 points) Experiment: Examine Nanog expression in an oct-4 homozygous knockout mouse, using in situ hybridization or immunostaining. This assumes the oct-4 KO exist. If not, we would have to generate it using homologous recombinant as discussed in class. [Would also accept RNAi instead of KO; would also accept work done in ES cells instead of in the embryo] [Some may answer that occt-4 is required for an ICM, in which case they may say that there is nothing to test in the "outcome section below. Reasonable answers based on this assumption will be accepted.] Expected outcome if Nanog expression depends on Oct-4 function: If Nanog expression requires oct-4 function, Nanog levels should be low/nonexistent Zoo 470 2008 Problem Set #3
Name:________________________________ Student Number:__________________ Page 3 3. Ali Hemati-Brivanlou and colleagues at Rockefeller University showed in 2004 that a pharmacological inhibitor of GSK-3 called BIO can induce human ES cells to maintain pluripotency in the absence of conditioned medium produced by mouse "feeder cells". a. Based on what you know, name at least three proteins that you expect to be in the nuclei of human ES cells treated with BIO. One of the three proteins you list should be a protein whose nuclear levels are known to be affected by inhibition GSK-3 activity (3 points) Protein 1 -catenin Protein 2: Nanog Protein 3: oct-4 [also accepted: Tcf/Lef. Stat3. Note: -catenin or Tcf need to be part of the answer] b. Brivanlou and colleagues deduced that the pathway affected by GSK-3 was involved in maintenance of pluripotency by comparing the expression of many thousands of genes in undifferentiated (pluripotent) human ES cells vs. differentiated ES cells. What technique did they use to do this? (1 point) Technique: DNA microarray ("gene chip") 4. Recall that mouse ES cells require leukemia inhibitory factor (LIF), produced by "feeder cells" to maintain pluripotency in culture. G-Q Zhao's lab at the University of Texas Southwestern Medical School showed earlier this year that one type of mouse feeder cells(called "C-STO" cells) were not as good at maintaining ES pluripotency than others, and set about trying to identify why (Hao et al., 2006). Hao et al (2006) identified 27 genes encoding secreted factors whose expression was reduced in C-STO cells. They then made transgenic cells expressing some of these genes, and examined their ability to support ES cells. a. Two of the genes they tested, when expressed in cultured cells, resulted in significantly lower levels of -catenin phosphorylation among ES cells when the cells were co-cultured. What type of secreted protein did these two genes likely encode? (2 points) Type of secreted protein: Wnts b. Hao et al (2006) found that the decrease in phospho--catenin caused an increase in transcription of a gene that encodes a transcription factor implicated in transducing LIF signals. What is this transcription factor? (2 points) Transcription factor: Stat3 c. Bonus question: The same transcription factor referred to in part c is known to affect cell migration events. Name a migration event in a non-human vertebrate that is regulated by this factor. (1 point) Cell migration event: Stat3 is known to control migration of cells during zebrafish gastrulation (see Fig. 11.11 of Gilbert) [if a citation is given for some other migratory event, this is acceptable] ...
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- Spring '08