Gene-targeting allows creation of knockouts and insertion of new genes. Gene targeting can have a similar outcome as transgenics, but the two methods involve a completely different methodology.
In gene targeting, a cell is exposed to an electric current which opens up pores in its membrane (elecotroporation). Subsequently, the cell uptakes genetic information from the environment and via homologous recombination acquires the allele of interest. A targeting vector containing flanking sequences homologous to the gene of interest but containing a mutation is constructed by routine molecular biology procedures. Then the targeting vector is transfected into ES cells in culture (electroporation), and the cells are then positively and negatively selected — and identified and confirmed by Southern Analysis — and then injected into the blastocyst cavity of developing mouse embryos.
Male chimeric 129/C57B/6 mice are formed, which are bred with female C57BL/6 mice to produce animals heterozygous for the targeted mutation; the F1 are then bred to produce homozygous F2 mice for the targeting vector integrated into the genome.
Selection markers are engineered into the homology region: the positive selection (+sel) marker is usually neomycin (confers G418 resistance) and ensures the cells were transformed at all; the negative selection marker is usually herpes simplex virus thymidine kinase (HSVtk) outside the region of homology.
HSVtk makes the cells sensitive to ganciclovir. While neomycin selects for cells with integration, the marker HSVtk selects for homologous recombination (since it is usually included in the genome only during random integration). Male ES cells are injected into a blastocyst (thus making it male) — the donor and recipient have different coat color alleles to make the male coat color (and thus gene targeted) chimeras easy to spot.
These are then mated with mice of the same strain as the blastocyst, generating heterozygous mice; these heterozygotes are then mated with each other to generate F2 homozygous gene-targeted mice.
The primary obstacle with gene targeting is that non-homologous recombination (aka random integration or non-targeted transformation) occurs much more frequently than homologous recombination (aka targeted transformation).
Positive/negative selection identifies rare homologous recombinants out of a population of cells with no and non-homologous recombination. First, positive selection kills all cells that were not transformed, leaving a population of random and targeted recombinants. Second, negative selection kills the random recombinants, so that only targeted (homologous) recombinants remain.
|Positive Selection||Positive selection identifies both targeted and non-targeted transformants; this can be accomplished by including an antibiotic-resistance gene on the transforming DNA, and then exposing the cell population to that antibiotic.|
|Negative Selection||After positive selection, negative selection is is performed to kill all non-homologous recombinants. Negative selection requires that the herpes simple virus thymidine kinase gene is included on the transforming DNA. Thymidine kinase makes cells die when exposed to the chemical gancyclovir, and is completely foreign to mammalian cells. Thus, cells undergoing random integration will carry both the foreign thymidine kinase gene and the homologous gene of interest. However, cells undergoing targeted recombination will only carry the gene of interest (not thymidine kinase) because there is no homologous region for the thymidine kinase gene to recombine. For this reason, gancyclovir will kill all non-homologous recombinants but preserve homologous recombinants.|
Knockouts, knockins and more
|CRE Integrases||CRE Integrases (causes recombination) are a family of site-specific enzymes which catalyze recombination. They delete segments of DNA flanked by LoxP sites (floxed) in experimental animals, and can generate animals with mutations limited to certain cell types (tissue-specific knockouts) or animals with chemically inducible mutations (inducible knockouts). There are transgenic lines of mice avaialble with tissue-specific or inducible Cre expression; breeding a floxed animal with a CRE-transgene animal allows researchers to create animals with genes that can be tissue-specific or induced.|
|LoxP Site||LoxP sites (locus of x-ing over) are 34bp sites where Cre has its effect. A DNA segment that is flanked by lox sites is floxed.|