Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells

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Abstract
We have analyzed the effects of linearizing vector DNA on the frequency and pathway of its recombination with the homologous chromosomal gene. The pSV2neo vector bearing a 4.3-kb fragment encoding the mouse immunoglobulin μ heavy chain constant (Cμ) region was cut either at sites within the Cμ segment or outside Cμ and then transferred to hybridoma cells bearing a mutant μ gene. The frequency of recombinant cells producing normal μ was then measured. For most cut sites, whether in regions of homology or of nonhomology, linearization of the transferred DNA enhanced the recombination frequency between the vector and chromosomal μ genes. When the vector was either uncut or cut at SacI in the region of homology, G418-resistant μ m+ recombinants were found to have integrated the vector by a single reciprocal homologous crossover; the enzyme site (SacI) used for cutting was present in the recombinants. By contrast, when the vector had been linearized at Pvul or SfiI in the region of nonhomology, vector integration involved nonhomologous crossovers, either between transferred DNA molecules or between transferred and chromosomal DNA, and the vector cut sites were absent in these recombinants. Some recombinants were found to have an unaltered as well as recombinant μ gene, suggesting that the nonhomologous recombination process might have involved sister chromatids.