Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice.

摘要

The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of 2 genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase. This generally requires 1-2 months of work for each construct. The single 'knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection(using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knock-out vector. This approach leads toinactivation of the gene of interest. An in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing. An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition. The DNA is then purified, and subsequently electroporated into bacteria. The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events. In the present study, this system was modified to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs. This approach led from genomic clone to completed construct ready for transfection in a matter of days. The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs, simplifying this technically demanding method.