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MIDTERM Name 1 MCDB 168 April 30, 2008 Name:________________________________________ ID#:__________________________________________ Section:_______________________________________ SCORE:____________ WRITE YOUR NAME ON EVERY PAGE WRITE CLEARLY, GRADERS GET UPSET IF THEY CAN'T READ THE ANSWER. PLEASE WRITE YOUR ANSWERS ONLY IN THE SPACE PROVIDED. Page 2 (15 pts) Page 3 (10 pts) Page 4 (13 pts) Page 5 (12 pts) Page 6 (11 pts) Page 7 (12 pts) Page 8 (7 pts) 1 Name 1) Which statement is true for mouse and/or human ES cells? Oct4 is an important regulator of their pluripotency (2.5 pts) a) Mouse ES-cells b) Human ES-cells c) Mouse and human ES-cells d) Neither Respond to LIF (leukemia inhibitory factor) in maintaining their pluripotent state (2.5 pts) e) Mouse ES-cells f) Human ES-cells g) Mouse and human ES-cells h) Neither Are typically derived from blastocysts that are generated through IVF (2.5 pts) i) Mouse ES-cells j) Human ES-cells k) Mouse and human ES-cells l) Neither 2) Primordial germ cells are (2.5 pts) a) Totipotent b) Multipotent c) Unipotent d) Pluripotent 1 point was given for "totipotent" 3) Unpaired (Upd) is an important component of (2.5 pts) a) Wnt signaling pathway b) JAK/STAT signaling pathway c) Notch signaling pathway d) BMP signaling pathway 4) Which of the following statements is FALSE? (2.5 pts) a) Egr1 is NOT required for hematopoietic stem cell self-renewal in adult b) Bmi1 is required for hematopoietic stem cell self-renewal in adult c) SCL is not required for hematopoietic self-renewal in adult d) Sox17 is required for hematopoietic stem cell self renewal in adult 2 Name 5) Define the following terms a) Blastocyst (3pts) The blastocyst is a developing embryo before implantation (around E3 in mice and E5 in humans) (1 point for timing) with two distinct cell types, the trophoblast (1) and the inner cell mass (1). b) Blastomere (3pts) A blastomere is one cell (1) from the 4-8 cell stage embryo (before blastocyst formation) (1) and before any specification has occurred and therefore it is totipotent (1). 6) By which assays would you test pluripotency of mouse and human cells (4pts)? IF for pluripotency markers, morphology of colonies, (Mouse and Human) (1pt) Embryoid body formation in culture to see development of all germ layers (Mouse and Human) (1pt) Teratoma formation in vivo to see development of all germ layers (Mouse and Human) (1pt) Chimeras, germline contribution or tetraploid complementation (Mouse only) (1pt) Additional: Expression of transcription factors, epigenetic regulation at promoters. 3 Name 7) Describe how embryonic stem cells, embryonic germ cells and embryonic carcinoma cells are derived (6pts)? ES cells are derived from the inner cell mass of the blastocyst. (2) EC cells are derived from teratocarcinomas (2) EG cells are derived from PGCs (2) 8) How does imprinting change during germ cell development (4pts)? During PGC migration (1) the cells undergo complete epigenetic erasure and global hypomethylation. This is when maternal and paternal imprinting (2) is erased and will only be reset (re-established) once the germ cell reaches either a female or male gonad. In other words, the re-established imprinting will be based on the sex of the offspring (1). If for example, the germ cell reaches a female gonad only the maternal imprinting will be set. 9) Explain the difference between therapeutic cloning and reproductive cloning? (3 pts) therapeutic cloning: SCNT is used to establish clones ES cells which are used to generate patient specific replacement cells for cell based therapy. (1.5) reproductive cloning: SCNT is used to generate an identical clone (living) of an individual. A cloned blastocyst is implanted and brought to term. Unethical in humans. (1.5) 4 Name 10) a) Describe a method that has been previously used to reprogram differentiated cells into a pluripotent state? (5 pts) Reprogramming by defined factors (IPS): Introduce a selection marker, either GFP under the control of Oct4 or Nanog promoter; or use the Fbx15 regulated neomycin resistance (1). Retroviral or lentiviral (1) introduction of Sox2, Oct4, cMyc and Klf4 (2) into adult fibroblasts. Culture on feeders (MEFs) in same growth conditions as ES cells to maintain pluripotency (1). Select pluripotent cells using GFP expression or neomycin resistance. OR SCNT: Somatic cell nuclear transfer that requires transplanting a nucleus from a differentiated cell (1) into an unucleated oocyte (2). Factors/molecules present in the cytoplasm of the oocyte (1) facilitate the reprogramming of the differentiated nuclei. Cloned blastocysts are used to generate cloned animals (e.g. cloning of Dolly was done using SCNT), or to establish cloned ES cell lines. (1) b) What advantages would there be to use IPS cells for stem cell based therapy rather than using EScells? (3 pts) Avoid ethical concern as there is no destruction of potential life. (1) Unlimited supply of adult fibroblasts that can be reprogrammed, whereas there is a limited supply of immune matched ES cells. (1) Availability of oocyte limits generation of patient specific ES cell lines. No immune rejection, if patient specific IPS cells are generated. (1) c) Suggest two reasons why IPS chimera mice are tumor prone, and what would you propose to do to improve this protocol in order to overcome this risk? (4 pts) The use of cmyc (oncogene), and reactivation of c-myc expression (1): Establish alternative protocols that do not require use of c-myc or other oncogenes, or use Cre-Lox system to excise coding region of oncogene The (1). issue of viral integration, which may activate on oncogene or inactivate a tumor suppressor via insertional mutagenesis (1): reprogram using transient protein, or RNA, characterize insertion sites before using cells for therapy. Screen small molecule, to find combinations of molecules to induce reprogramming. (1) 5 Name 11) Name a stem cell system where asymmetric division is clearly demonstrated and describe how this process is mechanistically regulated? (5pts) In the Drosophila testis (1), the hub (1), or niche, regulates the division pattern of germ line stem cells. Adherent interactions (cadherins) (1) between the hub cell and the stem cell and intracellular components (APC, beta-catenin, centrosome) (1) orient the mitotic spindle of the stem cell such that one cell remains in contact with the hub, whereas the other cell looses contact with the hub. Loss of interactions between the hub cell (which expresses Upd) and the distal daughter cell interrupt Jak/Stat signaling (1) required in stem cells, therefore promoting differentiation. This asymmetric, stereotypic pattern of division limits the number of potential germ line stem cells. Other answers describing other systems were given credit. 12) Describe the major sources of hematopoietic stem cells that are currently used in transplantation therapy, and describe at least two advantages or disadvantages in using each source. (6pts) Bone marrow Advantages: most experience, fast hematopoietic recovery Disadvantages: few donors available, risk for viral pathogens in blood, risk for graft vs. host disease (2) Umbilical cord Advantages: cord blood banks, less immunogenic, lower risk of blood borne pathogens Disadvantages: low number of HSCs for adult patients, slow neutrophil recovery (2) Mobilized peripheral blood Advantages: autologous and allogenic transplants possible, accessible, less invasive Disadvantages: fewer cells than bone marrow, time consuming (2) 13) During your first year of graduate research at Dingle Berry University you perform a genetic screen in zebrafish, to identify mutants that affect hematopoiesis. You uncover a gene called vampire, and create a mouse knockout of vampire. Unfortunately the knockout is lethal. You hypothesize vampire is critical in mouse developmental hematopoiesis. a) State the organs you would examine to determine if there is an embryonic hematopoietic defect, and what would you look for to define the nature of the defect (6pts)? 6 Name Developmental hematopoiesis occurs in discrete anatomical sites and at different developmental time points. I would look in the yolk sac for primitive red blood cells and transient progenitors. I would also examine the placenta and AGM for evidence of stem cell generation. To see if HSC expansion and differentiation are affected, I would look in the placental labyrinth (expansion) and fetal liver (both) . I would use a combination of colony forming assays, histology, FACS and transplantations to determine the nature of the defect. (1.5 pt for describing analysis of each 4 organs) b) How do you propose to bypass the embryonic lethality so that you could study function of vampire in adult hematopoiesis? (3pts) I would create a conditional knockout animal for vampire using a loxP flanked allele (1). In these animals, both alleles of vampire would be flanked by loxP sites. I would breed theconditional strai with another mouse strain that carries Cre recombinase gene (1) under the control of a promoter that facilitates excision after embryonic development ( e.g.inducible Mx-responsive promoter) (1). Thus, I could treat these animals with polyI-polyC and induce excision in adulthood and then study the effects. c) How would you test if vampire is required for HSC differentiation? (3pts) After generating a conditionally targeted mouse model, I would examine peripheral blood of mutants by FACS (1) for markers of different blood lineages (e.g myeloid, erythroid, lymphoid) (1) and compare the profile to WT. I would perform colony assays for bone marrow (if using conditional mouse model, or fetal hematopoietic organs if using the conventional knockout) in methylcellulose, and compare the number and differentiation potential of colonies produced in both WT and vampire animals. (1) d) How would you test if vampire is required for HSC self-renewal? (4 pts) The most reliable test for self-renewal is serial transplantation (2). To do this experiment, I would harvest bone marrow from CD45.1 animals (or any marker that allows distinguishing between different individuals) and isolate HSCs using FACS. I would then inject these cells along with support cells from a CD45.2 animal into an irradiated, CD45.1/CD45.2 recipient. I would wait 16 weeks, then harvest bone marrow and transplant again to irradiated animals. After another 16 weeks, I would examine both the bone marrow and peripheral blood for CD45.1 (donor marker) cells; lack of these would indicate self-renewal defects in vampire HSCs. (description 2 pt) Other answers that were feasible were given credit. e) If you find out that vampire functions in HSC self-renewal, propose an experiment you could assess the mechanism of its action? (3pts) I would perform microarray analysis of HSCs comparing vampire mutants to WT animals. Analysis would focus on genes known to function in self-renewal (e.g. Bmi1) and differentiation. I could also perform ChIP-chip using an anti-vampire antibody to determine which genes vampire regulates (if it's a transcription factor). Other answers that were feasible were given credit. 7 Name 8

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