Disruption of the Gi2α locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair

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Abstract
We have used an insertion vector-based approach to target the Gi2α gene in AB-1 embryonic stem cells. 105 bp located 0.8–0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineeredNot I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained (‘hit’ step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy, 2-fluoro-β-d-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination (‘run’ step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified ‘hit and run’ strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated Gi2α allele is also demonstrated.