Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper

We have developed a method to clone genomic DNA selectively into a yeast-ba cterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A g ene-specific pClasper targeting vector was constructed with small recombino genic ends (500 bp) derived from flanking sequences of the genomic region t o be cloned. Linearized, recombinogenic pClasper targeting vector and nativ e genomic DNA mere co-transformed into yeast. The gene of interest is selec tively cloned by recombination between the recombinogenic ends in the targe ting vector and homologous regions in the genomic DNA. Here we demonstrate direct cloning of a stably integrated Hoxc8-LacZ-Ura3 reporter gene constru ct from a mouse embryo fibroblast cell line and single-copy genes from tota l human genomic DNA. The frequency of capture of the recombinant insert was 0.05-3% of transformants. In contrast to previous reports, we were able to clone genomic DNA directly with a vector containing yeast autonomous repli cating sequences. This approach provides a powerful method with which to cl one and modify genes precisely for functional analysis. (C) 1999 Academic P ress.