DISRUPTION OF THE G(I2)ALPHA LOCUS IN EMBRYONIC STEM-CELLS AND MICE -A MODIFIED HIT AND RUN STRATEGY WITH DETECTION BY A PCR DEPENDENT ON GAP REPAIR

We have used an insertion vector-based approach to target the G(12)alp ha gene in AB-1 embryonic stem cells. 105 bp located 0.8-0.9 kb upstre am of a disrupting Neo marker in exon 3 were deleted and replaced with an engineered Not I site, that served to linearize the vector. The 10 5 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homol ogous recombination. Both target conversion and vector insertion event s were obtained ('hit' step). Clones that had inserted the entire targ eting vector were taken into FIAU (1-[2-deoxy, 2-fluoro-beta-D-arabino furanosyl]-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 intrachro mosomal recombination ('run' step). Additional selection in G418 reduc ed the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more spec ific selection of the desired intrachromosomal recombination event in tissue culture. This modified 'hit and run' strategy represents a nove l approach for vector design and the use of the polymerase chain react ion to detect targeting. It may be particularly useful for targeting g enes that display a low frequency of homologous recombination. Germ li ne transmission of the mutated G(12)alpha allele is also demonstrated.