1. MCDB 4650 Mouse Developmental Genetics and Genetic Engineering

2. In mammalian blastocyst or gastrulation stage embryos (mark all that are true), a) the inner cell mass (ICM) will give rise to the epiblast and the trophoblast will give rise to the hypoblast of the germinal disc. b) cells of the ICM are committed to specific embryonic fates. c) if ICM cells from a donor blastocyst are injected into a recipient blastocyst, these cells can contribute to any tissue of the developing embryo. d) cells from both the ICM and the trophoblast participate in gastrulation. e) the primitive streak forms on the future dorsal side of the germinal disc. f) the primitive streak begins near the posterior end of the germinal disk and moves anteriorly.

3. In the mouse early embryo, blastomeres become committed (i.e. no longer totipotent) at the A) 4-cell stage B) 8-cell stage C) morula stage D) blastocyst stage E) after the blastocyst stage

4. If you inject ES cells homozygous for a dominant marker, e.g. black coat color, into a blastocyst from a white mouse, and then mate the resulting animal to another white mouse, the chances of obtaining any all-black progeny will be A) Zero, because the injected cells cannot become part of the germ line. B) Zero, because any embryo fertilized with gametes derived from the ES cells will be variegated (patched black and white). C) Predictable from the ratio of injected ES cells to host ICM cells in the injected blastocyst. D) Unpredictable.

5. If the construct inserts by HR, recombination will occur A) in the yellow (vector) regions only. B) in the blue and yellow regions only. C) in the red regions only. D) in all regions with equal probability. TK Neo r YFG DNA construct Vector

6. You transfect ES cells from a black mouse (dominant marker) with this construct : Following transfection, the culture will include A) cells that have lost the transgene without inserting it B) cells that have inserted the transgene by homologous recombination at the YFG locus C) cells that have inserted the transgene at another locus by end-joining to a nicked strand followed by DNA repair. 1) In non-selective growth medium (no neomycin or gancyclovir) the most frequent class of cells will be ___. TK Neo r YFG Vector

7. You transfect ES cells from a black mouse (dominant marker) with this construct : Following transfection, the culture will include A) cells that have lost the transgene without inserting it B) cells that have inserted the transgene by homologous recombination at the YFG locus C) cells that have inserted the transgene at another locus by end-joining to a nicked strand followed by DNA repair. TK Neo r YFG Vector 2) In medium containing gancyclovir alone, the most frequent class of cells will be ___.

8. TK Neo r YFG Vector A) cells that have lost the transgene without inserting it B) cells that have inserted the transgene by homologous recombination at the YFG locus C) cells that have inserted the transgene at another locus by end-joining to a nicked strand followed by DNA repair. 3) In medium containing neomycin alone, the most frequent class of cells will be ___.

9. TK Neo r YFG Vector A) cells that have lost the transgene without inserting it B) cells that have inserted the transgene by homologous recombination at the YFG locus C) cells that have inserted the transgene at another locus by end-joining to a nicked strand followed by DNA repair. 4) In medium containing neomycin and gancyclovir, the most frequent class will be ___.

10. You inject embryonic stem cells (from a black mouse) that have undergone homologous recombination at the YFG locus, into blastocysts obtained from a mated white mouse and reimplant them into pseudopregnant white females. When she gives birth to offspring (Po mice): A) all will be variegated (both black and white patches of coat color). B) a few may be white, but the majority will be variegated. C) some will be white, some will be variegated.

11. You mate a variegated (chimeric) female mouse to a white male, and examine their offspring (F1) for coat color and the presence of the knocked out YFG. Which of the following statements is true? (write out for yourself the kinds of gametes she makes) (Remember: the white mice are homozygous black –/black –; black coat color is dominant, and the black gene is on a different chromosome from YFG). A) All the black offspring are homozygous for the transgene (YFG-/YFG-) B) Some of the black offspring are homozygous for the transgene (YFG-/YFG-) C) All of the black offspring are heterozygous for the transgene (YFG-/YFG+) D) All of the black offspring are homozygous normal (YFG+/YFG+) E) Some of the black offspring are heterozygous for the transgene (YFG-/YFG+)

12. A transgenic mouse in which a randomly inserted transgene is expressed in every cell of the adult can be made by a) injection of the transgene into the male pronucleus in a fertilized mouse egg and reimplanting the resulting embryo in a foster mother. b) introduction of the transgene with no selection into cultured ES cells and injection of the transformed ES cells into a host blastocyst stage embryo, which is then reimplanted. c) introduction of the transgene into cultured ES cells and screening for incorporation by homologous recombination before injection of the transformed ES cells into a host blastocyst stage embryo and reimplantation.

13. A transgenic mouse in which a gene of interest is knocked out in all cells of the animal can be made by constructing a defective cloned copy of the gene carrying a selectable marker and a) injecting the construct into the male pronucleus in a fertilized mouse egg and reimplanting the resulting embryo in a foster mother. b) introducing the construct with no selection into cultured ES cells, injecting the transformed ES cells into host blastocyst stage embryos which are then reimplanted, and interbreeding the resulting progeny. c) introducing the construct into cultured ES cells and screening for incorporation by homologous recombination before injecting the transformed ES cells into host blastocyst stage embryos which are then reimplanted, and interbreeding the resulting progeny.

14. Which of the following is/are true? Injection of a cloned mouse transgene into one pronucleus of a fertilized mouse egg results in a) integration by recombination of that piece of DNA at its homologous chromosomal sites. b) random integration of the DNA into any chromosome. c) self-recombination of the DNA into a large extrachromosomal array that does not integrate. d) presence of that DNA in every cell in the body of the resulting mouse. e) a chimeric mouse, with that DNA present in some cells but not in others.

15. Suppose you are studying a gene that is required for neuronal development in mouse, and is expressed throughout the central nervous system (CNS). You have developed two transgenic mice: Strain 1 is homozygous for the reporter construct diagrammed below, comprised of a lacZ gene driven by an actin promoter that is expressed in all cells, flanked by two loxP sites. Strain 2 is homozygous for the Cre recombinase gene driven by the promoter for the neuronal gene you are studying. All tissues of the Strain 1 mice stain blue with an X-gal stain for  -galactosidase activity. If you mate mice from Strain 1 and Strain 2, and analyze tissues of the resulting progeny for staining with X-gal, you will find that (choose the one most likely answer): a) All tissues stain blue. b) Only the CNS stains blue. c) All tissues except the CNS stain blue. d) None of the tissues stain blue. Strain 1 transgene

16. What does transgenic mean? a.The same thing as chimeric or mosaic b.Some cells contain a piece of foreign DNA c.All cells contain a piece of foreign DNA d.Some cells contain a mutation in a specific gene

17. How do you generate a line of homozygous mutant mice? a.Mutagenize a bunch of mice and screen through their progeny for phenotypes that interest you b.Knock out a specific gene using RNAi c.Engineer the permanent removal of a specific sequence of DNA from the genome

18. Will a chimeric transgenic mouse always yield transgenic progeny? a. Yes, always b. No, not necessarily

19. Two dozen lethally irradiated mice survive after injection with a small amount of mixed bone marrow cells from two donors, one of which carries an easily assayable DNA marker. The surviving mice have functioning immune systems and all the normal types of blood cells. In any given mouse, either all cell types carry the marker or none of them do. When sacrificed and dissected, 21 of these mice are found to have only one spleen nodule, containing myeloid cells but no lymphoid cells. Which one or more of the following could you conclude to be true? a) A spleen nodule arises from a single hemopoietic stem cell. b) All blood cell types can arise from a common progenitor stem cell. c) Myeloid and lymphoid cells must arise from different precursor stem cells.

20. Recessive mutations in either the kit gene or the steel gene cause similar phenotypes that include severe anemia. The corresponding gene products were suspected to involve interactions between hematopoietic stem cells and the microenvironments that induce them to differentiate correctly. Experiments to test this possibility showed that when young lethally irradiated steel mutant mice are injected with bone marrow cells from unirradiated kit mutant mice, the mice die. However, when the converse experiment is done, (irradiated kit mutant recipients, injected with bone marrow cells from an unirradiated steel mutant), the mice survive. The most likely functions of the Steel and Kit gene products are: 1) Steel is a receptor on cells in the hematopoietic microenvironment; Kit is a ligand secreted by hematopoietic stem cells. 2) Steel is a ligand secreted by cells in the hematopoietic microenvironment; Kit is a receptor on hematopoietic stem cells. 3) Steel is a receptor on hematopoietic stem cells; Kit is a ligand secreted by cells in the hematopoietic microenvironment. 4) Steel is a ligand secreted by hematopoietic stem cells; Kit is a receptor on cells in the hematopoietic microenvironment.