ps3_2008 - Zoo 470 2008 Problem Set#3 Problem Set#3 ES Cell...

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Unformatted text preview: Zoo 470 2008 Problem Set #3 Problem Set #3 ES Cell Potency Zoology 470 Spring 2008 20 points total Page 1 Problem Set Guidelines 1. Due date: This problem set is due by 5 pm on Monday, May 5, 2006. It may be submitted either in class or to the mailbox of the course TA, Allison Lynch, in the Zoology Research Building. 2. Sources: You may use any sources at your disposal to answer the following questions. Legitimate sources include classmates, knowledgeable friends and colleagues, written documents, and any other scientific resources you find useful. If you work with other classmates on this problem set, we ask that you list the other students with whom you worked to answer these questions. Although you may discuss these questions as part of a group, you are expected to answer the questions as an individual. If you believe that published references will help you answer these questions, you may cite those references. However, citation of additional references is not required, nor is it expected. 3. Answering the questions: This problem set is designed to be answered concisely. Brief but complete answers should be written in the space provided. If you find it helpful, feel free to include diagrams in your answers. You need only turn in your answers on pages 2 & 3. 4. Necessary information: All of the information and techniques needed to answer the questions on p. 2-3 have been presented in class, or are to be found in Gilbert's Developmental Biology. Note: some techniques may require you to review information that was covered on previous exams. You may also wish to consult several primary papers: Nanog: Chambers et al (2003). Cell 113:643-55; Mitsui et al (2003). Cell 113:631-42 GSK-3: Sato et al (2004). Nature Medicine 10:55-63. C-STO cells: Hao et al (2006). Developmental Biology 290:8191 Problem Statement Attempts are currently underway to identify factors that will allow human embryonic stem (ES) cells to maintain their pluripotency in the absence of mouse "feeder cells". Studies using the mouse as a model system have shown that in addition to Oct-4, Nanog (named after a mythical Celtic land of the ever-young) and other transcriptional regulators are important in maintaining pluripotency. The questions on pp. 2-3 are designed to explore how these proteins interact to maintain pluripotency. 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: 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) 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) d. Where within the cells that produce Nanog protein would you expect it to be found? (1 point) Location within cells: 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: Expected outcome if Nanog expression depends on Oct-4 function: 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: Protein 2: Protein 3: 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: 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: 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: 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: ...
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