We propose to use phenotypic selection to study recombination between endogeneous and exogeneous genes in mammalian somatic cells (targeted recombination). Our experiments will entail analysis of human cell lines that either contain a single functional allele for the adenine phosphoribosyltransferase (APRT) gene (hemizygous cells), or that lack functional APRT genes altogether, (APRT-cells). Hemizygous cells will be used in a marker inactivation assay whereby homologous recombination of the lone endogeneous APRT gene with a plasmid introduced by transfection leads to the total loss of APRT activity. APRT- cells will be used in a marker rescue assay whereby homologous recombination between a mutated endogeneous allele and a rescue-plasmid restores APRT function. These selective assays of targeted recombination will be used to study (i) the frequency and modes of information exchange between chromosomal DNA and DNA molecules introduced by transfection, (ii) structural and functional features that influence the recombinogenicity of chromosomes as recipients of exogeneously supplied DNA and as substrates for genomic rearrangement, (iii) the mechanism of recombination in chromosomal DNA.
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| Stringer, J R; Kuhn, R M; Newman, J L et al. (1985) Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells. Mol Cell Biol 5:2613-22 |
