Work

Rapid Assay for Extrachromosomal Homologous Recombination in Monkey Cells

Year: 1985

Type: article

Abstract: Most of the recombination assays based on the regeneration of selectable marker genes after transient infection or stable integration of DNA into mammalian cells are time consuming. We have used plasmids containing two truncated but overlapping segments of the neomycin resistance gene to rapidly quantitate and characterize the time course of extrachromosomal homologous recombination of DNA transfected into monkey COS cells. By transiently infecting cells with these recombination substrates, extracting Hirt DNA after 1 to 4 days, and transforming recombination-deficient Escherichia coli, we have shown that recombination between direct repeats occurs at frequencies of 1 to 4%. We have also used Southern blot analysis to directly characterize the recombination of this DNA in COS cells and to demonstrate that double-strand breaks in the region of homology increase recombination frequencies 10- to 50-fold.

Source: Molecular and Cellular Biology

DOI: 10.1128/mcb.5.3.529

Authors: J Rubnitz, S Subramani

Institution: University of California San Diego

Language: English

Topic: DNA Repair Mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: Searle Scholars Program, National Institutes of Health, National Institute of General Medical Sciences

Award: G712794427