L14-15: Genetic Manipulation in Animals Flashcards
Creation & Applications of Transgenic Animals
Gene function and development: Transgenic drosophila, transgenic frogs and transgenic fish
Bioreactors: Transgenic sheep, transgenic pigs and transgenic cows
Biomedical research: Transgenic mice
Transgenic Mice
Zygote: take stud mice and get female mice pregnant =>produces zygote
- Inject into pronucleus
- Zygote become blastocyte
- Inject some into pseudopregnant mice (think they are pregnant, have the same hormonal levels)
Exogenous DNA =>transfection of primordial germ cells =>sperm&oocyte
ES cells=>cell transfer into blastocyte
Transgenic Animals
Produced by DNA transfer into totipotent or pluripotent embryo cells i.e., fertilized oocytes or pre/post-implantation embryos
Integration in fertilized oocytes produces fully transgenic animal
Integration at a postzygotic stage produce mosaic animal
Step 1 of Transgenic animals:
Pronuclear injection
Superovualted females are mated and sacrificed the next day.
Fertilized oocytes are microinjected with DNA using micromanipulator (microinjection pipette).
Surviving zygotes are re-implanted into the oviduct of foster females
Step 2 of Transgenic animals:
Transfer into embryos
Infection of preimplantation embryos with retrovirus or infection of early post-implantation embryos result in mosaic offspring
Step 3 of Transgenic animals:
Embryonic stem cells provide a route for germline genetic change
- Mouse ES cells are derived from the inner cell mass of 3.5- 4.5 days old embryos.
- ES can be cultured retaining their totipotency.
- ES cells can give rise to all tissues including germ cells, if placed back into a host blastocyst and reimplanted in pseudopregnant mouse.
- The embryo is a chimera. If the two strains of cells are derived from mice with different coat colors then the offspring can be easily distinguished.
- Most important advantage of using ES cells: the desired genetic modification can be confirmed in culture.
- The targeting vector usually has a neo gene which enables positive selection for cells that were successfully targeted.
- Gene targeting by homologous recombination allows for a selective alteration of a single predetermined gene
Transgenic Animals: Applications
- Study of gene expression and its regulation, i.e., reporter constructs –lacZ
- Gene function by targeted gene inactivation.
- Investigating dosage effects and ectopic expression, i.e., over-expression of transgene
- Cell lineage ablation: tissue-expression of toxins such as ricin.
- Investigating gain of function: ex: sry transgene expression in female embryos to produce male offspring.
- Modeling human disease: insertional inactivation or insertion of mutants.
Innovations in Transgenic Technologies (3)
- Inducible promoters: tetracycline-regulated or Tamoxifen-inducible.
- TetR added to tetO =transgene off; remove TetR w doxycycline =on
- Hsp90 attached =inactive; tamoxifen removes Hsp90 =active - YAC transgenes: to study human genes under expression of its promoter and to study large genes.
Application: production of human antibodies in mouse by transferring human YACs containing large segments of human heavy and kappa light chain immunoglobulin loci - Transchromic animals: microcell-mediated chromosome transfer allowed transfer of whole human chromosomes into mouse ES cells
ES Cells in Gene Targeting
- Gene targeting by homologous recombination: gene targeting= artificial site directed in vivo mutagenesis
- Homologous recombination b/w introduced gene and its chromosomal homolog. H.R. is rare in mammalian cells. Frequency of H.R. events is increased if flanking sequence homology is very high (isogenic sequences).
- To identify desired H.R. event, targeting vector should contain marker gene (neo) and can be introduced by electroporation into ES cells.
- Insertional vectors vs replacement vectors
Site Specific Recombination Systems, Cre-LoxP
Knock-outs
Tissue and cell type specific knock-outs: conditional KO →some genes are vital for early development.
It involves replacing part of the endogenous gene by a gene segment flanked by LoxP sequences.
Mice carrying this targeted mutation are mated with mice which carried a Cre recombinase transgene under the control of a tissue-specific promoter.
Tissue and cell type specific gene activation:
It involves selective activation of a gene in certain cells… How?
- Mouse with target locus A flanked w LoxP sites crossed w transgenic mouse w Cre genes linked to tissue-specific promoter of interest
- Offspring w floxed target plus Cre transgene –tissue or cell-specific deletion of A at target locus
o Loop and LoxP seq sites line up –remove A and leaves 1 LoxP site
Knock-Ins (2)
1) cDNA
o replaced exon1 with targeted insertion with involves cDNA (has its own polyadenylation sites but also its own promoter)
2) Reporter
o replace 1 allele w lacZ
Chromosome Engineering
It involves integrating loxP sites at the desired chromosomal locations and subsequently Cre expression is used to mediate a selected chromosomal re-arrangement.
- 2 loxP sites in diff chromosomes causes recombination which causes cross of chromosome arms
- loxP facing the same direction =deletion
- if facing each other =inversion
Large Scale approach to study animal gene function
Exposure to radiation or potent chemical mutagens such as ethylnitrosurea (ENU) or ethyl methylsulfonate (EMS).
Males are exposed and their progeny is screened for phenotypic abnormalities. The approach is useful but requires positional cloning to determine the DNA legion.
Insertion of a foreign DNA seq. presents an advantage because it leaves a tag at the site of mutation. It involves random integration (by nonhomologous recombination).
Insertion of reporter transgenes which lack some components required for expression = Gene Trapping. Avoids all the noncoding insertions.
Gene Trapping
Alter gene with splice acceptor sequence =>B-galactosidase
And polyadenylation signal =>neomycin phosphotransferase
Creation of Animal Models of Diseases
Allow for examination of the pathophysiology of disease.
Allow for testing potential therapies.
Some originated spontaneously; others were made by gene targeting.
Differences in disease phenotype b/w human and mice lead to insights on potential therapeutic mechanisms. TSD model.
