Week 1-2

Question 1

Cause of death in human embryo prior to E8

Answer 1

preimplantation loss
peri-implantation failure
death at gastrulation

Question 2

preimplantation loss

Answer 2

disruption of basic cellular functions no blastocyst formed

Question 3

peri-implantation failure

Answer 3

no connection between trophoblast and the decidua\

Question 4

death at gastrulation

Answer 4

failure to form the definitive germ layers failure to elaborate mesoderm

Question 5

Death during organogenesis (~E10-14)

Answer 5

1. blood flow in the yolk sac
2. failure to establish a chorio-allantoic placenta
3. trophoblast defects
   The trophoblast is an extra-embryonic lineage required for placental function.

Question 6

Death in early fetal period (~E14-19)

Answer 6

heart defects

liver hematopoiesis

Question 7

Developmental disturbances that lead to death of the embryo

Answer 7

1. failure to establish and maintain a vascular circulation
2. failure to make the transition from yolk sac to liver hematopoiesis
3. abnormal interaction with maternal environment (implantation, yolk sac vascular circulation, formation of chorio-allantoic placenta)

Question 8

Organs and systems with no survival value in utero

Answer 8

central nervous system gut
lungs
urogenital system musculoskeletal system
- abnormalities in these systems can often lead to perinatal or early postnatal lethality…

Question 9

What do you need to build a targeting vector specific to your gene?

Answer 9

1. homologous DNA; need a region of homology
2. positive selection for vector incorporation
3. negative selection against random integration events
4. screening strategy for homologous recombination events

Question 10

negative selection against random integration events

Answer 10

Examples: tk, DTA; facultative
tk is a viral gene, if cell produces it cell is okay, but if you put drug in the cell will eat drug and die
DTA is a toxin

Question 11

what are drug markers for positive selection for vector incorporation

Answer 11

neo, puro, hyg, zeo,…

drug markets __mycin

Question 12

How to find homologous DNA

Answer 12

library screening
genomic PCR (easy with genome sequence available!)

Question 13

How to find homologous DNA

Answer 13

library screening
genomic PCR (easy with genome sequence available!)

Question 14

How to do Gene targeting and chromosome engineering in the mouse genome

Answer 14

1. Build a targeting vector specific to your favourite gene.
2. Produce a mutation in ES cells by electroporating the construct that will target a change to one allele of this gene following homologous recombination.
3. Isolate clonal lines of correctly targeted ES cells > confirm mutation.
4. Produce ES cell chimeras by injecting the correctly targeted ES cells into a host mouse blastocyst or by doing a morula aggregation.
5. Breed the male chimeras to recover offspring carrying the targeted mutation through germ line transmission of the ES genome.
6. Analyse the results of the mutations in heterozygous and homozygous mice by breeding: phenotypic analysis.