Transgenics Flashcards
What are the advantages and disadvantages of southern blot?
Good - can detect insertion and rearrangement of transfers and copy number
Bad - requires lot of DNA (10-50 micrograms)
Long and complex technique
No info on site of integration
Describe the method of southern blotting
Genomic DNA extraction Restriction digest Fractionate by agarose GE Denature DNA transfer to carrier membrane Block membrane (prevent NS binding) Hybridise with labelled probe Wash to remove NS binding
Describe northern blotting
Extract mRNA Fractionated by GE Transfer to carrier membrane Block membrane to prevent NS binding Hybridise with labelled probe Wash to remove NS binding Detect by autoradiography
What are the differences between donors/clones?
Clones - mitochondria is mix of original oocyte/diploid somatic cell
Telomeres shorten during SC proliferation , so are shorter than donor
describe somatic nuclear transfer in sheep
cultured foetal cells transfected with foreign DNA - integrated clones selected
diploid nuclei introduced to enucleated oocytes
oocyte cytoplasm controls immediate post fertilisation development
nuclei initially transcriptionally quiescent
describe the background of somatic nuclear transfer
use of somatic cell rather than embryo
Wilmut et al (Roslin) achieved SNT in sheep (Dolly)
nuclear transfer into fertilised egg cells - oocyte reimplanted into foster ewes
low success rate but dolly born (1/434 oocytes)
describe similarities and differences between somatic nuclear transfer and the ES cell route
similarity - starting point of both = introduction of foreign DNA, which permits selection
favourable difference - diploid nucleus introduced directly into recipient oocyte, supplying genome of whole animal (no further breeding needed)
unfavourable = method remains inefficnent
describe PCR and its advantages / disadvantages
extract genomic DNA (<1 ug) and design pair of f/r primers to amplify transgene
perform PCR and analyse with agarose GE
good - detect transgene integration - small amount required (<10 ng DNA), rapid and easy
bad - prone to contamination - no info on site of integration - hard to determine copy number
describe pro nuclear micro injection
hormonal induction of super ovulation - PMSG then 48h later HCG= 1 cell embryos recovered and held in micro suction pipette
DNA introduced into male pronucleus through glass needle
development of embryo in 2-4 cell stage- to recipient dam
in recipient dams, pseudopregnant recipients synchronised by mating with vasectomised males
describe main stages of pro nuclear micro injection and its problems
super ovulation - PMSG and HCG
DNA -> male pronucleus
synchronised mating with vasectomised males in recipient dams
insertion site + copy number = random, cannot be repeated so characterisation must be carefully chosen
describe the main findings of henderson et al 2002 (P450)
Deletion of NADH ferrihemoprotein reductase in liver = ablation of hepatic microsomal P450 DNA
hepatic cpr null mice could no longer break down cholesterol, could still breed, so p450 hormone metabolism not essential for fertility
describe the main findings of otto et al 2003 (Cy P450)
C P450 dependent mono oxygenase system catalyses metabolism of xenobiotics/endogenous compounds
investigated role in embryogenesis - inactivated system through deletion of cpr e- donor
mouse embryos homozygous for deletion died in early-mid gestation - defects in cells where cpr expressed - brain limbs etc
describe main findings of okabe et al 1997 (GFP)
produced transgenic mouse lined with E GFP
insert wt GFP -> pCAGGS = transgenic mice - then add E GFP
green F = green eggs - maternal E GFP already active in unfertilised egg
Produce source of green reimplantation stage embryo - used to produce chimeric mice by injection/aggregation of non green ES cells - allow early ID of ES tissue
study tumorigenesis - implant non green tumour cells into green mice
describe main steps of ES cell modification
derive pluripotent ES cells from ICM blastocyst
selective killing of non trans/diff cells - oct3/4- also controls neo r gene
transfected into blastocysts and cultured in neomycin
selective killing of non trans/diff cells
alter target gene by homologous recombination (similar strands of dsDNA)
microinjection of transfected ES cells into blastocysts to produce germ line chimeras - introduced to recipient dams
3 types of gamete produced when mating chimeras with normal mice - derived from host blastocyst, from ES cell, but not GM/or GM
what is a transgene?
a recombinant DNA molecule including a regulatory element to control transcription and a structural element composed of DNA for gene synthesis
building one = empirical process, ectopic expression is common
