Week 1-2 Flashcards
Transgenesis, preimplantation, stem cells, and chimeras
Cause of death in human embryo prior to E8
preimplantation loss
peri-implantation failure
death at gastrulation
preimplantation loss
disruption of basic cellular functions no blastocyst formed
peri-implantation failure
no connection between trophoblast and the decidua\
death at gastrulation
failure to form the definitive germ layers failure to elaborate mesoderm
Death during organogenesis (~E10-14)
- blood flow in the yolk sac
- failure to establish a chorio-allantoic placenta
- trophoblast defects
The trophoblast is an extra-embryonic lineage required for placental function.
Death in early fetal period (~E14-19)
heart defects
liver hematopoiesis
Developmental disturbances that lead to death of the embryo
- failure to establish and maintain a vascular circulation
- failure to make the transition from yolk sac to liver hematopoiesis
- abnormal interaction with maternal environment (implantation, yolk sac vascular circulation, formation of chorio-allantoic placenta)
Organs and systems with no survival value in utero
central nervous system gut
lungs
urogenital system musculoskeletal system
- abnormalities in these systems can often lead to perinatal or early postnatal lethality…
What do you need to build a targeting vector specific to your gene?
- homologous DNA; need a region of homology
- positive selection for vector incorporation
- negative selection against random integration events
- screening strategy for homologous recombination events
negative selection against random integration events
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
what are drug markers for positive selection for vector incorporation
neo, puro, hyg, zeo,…
drug markets __mycin
How to find homologous DNA
library screening genomic PCR (easy with genome sequence available!)
How to find homologous DNA
library screening genomic PCR (easy with genome sequence available!)
How to do Gene targeting and chromosome engineering in the mouse genome
- Build a targeting vector specific to your favourite gene.
- Produce a mutation in ES cells by electroporating the construct that will target a change to one allele of this gene following homologous recombination.
- Isolate clonal lines of correctly targeted ES cells > confirm mutation.
- Produce ES cell chimeras by injecting the correctly targeted ES cells into a host mouse blastocyst or by doing a morula aggregation.
- Breed the male chimeras to recover offspring carrying the targeted mutation through germ line transmission of the ES genome.
- Analyse the results of the mutations in heterozygous and homozygous mice by breeding: phenotypic analysis.