NOVEL LACZ-BASED RECOMBINATION VECTORS FOR MAMMALIAN-CELLS

We have constructed two sets of Escherichia coli lacZ-based vectors fo r use in studies of general mitotic recombination, both in somatic mam malian cells grown in culture and in transgenic animals. The vectors u se two mutant copies of the E. coli lacZ gene as their recombination s ubstrates and contain a neo gene for selection of stable transformants . In one vector, pLrec, an SV40 promoter drives lacZ, while the other vector, pArec, utilizes a human non-muscle beta-actin promoter for lac Z expression. Gene conversions, unequal sister chromatid exchanges and reciprocal exchanges between the two lacZ genes result in expression of beta-galactosidase, which can be detected in situ by histochemical staining. These vectors yield rates and frequencies of mitotic intrach romosomal recombination in human and rodent cell lines which are simil ar to rates reported using conventional recombination vectors. Molecul ar analysis of recombinational events involving the lacZ-based vectors can be carried out on genomic DNA isolated from clonally expanded pop ulations and individual LacZ(+) cells and cell clusters can be analyze d using PCR amplification. These reporter gene-based vectors may facil itate the study of recombination in cells with limited proliferative c apacities, allow for analysis of both products of an unequal sister ch romatid exchange, and permit in situ detection of recombination in the tissues of transgenic animals.