Novel lacZ-based recombination vectors for mammalian cells.
We have constructed two sets of Escherichia coli lacZ-based vectors for use in studies of general mitotic recombination, both in somatic mammalian cells grown in culture and in transgenic animals. The vectors use two mutant copies of the E. coli lacZ gene as their recombination substrates 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 lacZ 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 intrachromosomal recombination in human and rodent cell lines which are similar to rates reported using conventional recombination vectors. Molecular analysis of recombinational events involving the lacZ-based vectors can be carried out on genomic DNA isolated from clonally expanded populations and individual LacZ+ cells and cell clusters can be analyzed using PCR amplification. These reporter gene-based vectors may facilitate the study of recombination in cells with limited proliferative capacities, allow for analysis of both products of an unequal sister chromatid exchange, and permit in situ detection of recombination in the tissues of transgenic animals.
Herzing
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Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.
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D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005785:Gene Conversion; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D051379:Mice; D008822:Mice, Transgenic; D008938:Mitosis; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012854:Sister Chromatid Exchange; D014170:Transformation, Genetic