The Mechanism of Mammalian Gene Replacement Is Consistent with the Formation of Long Regions of Heteroduplex DNA Associated with Two Crossing-Over Events

Abstract

In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin μ-δ region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCμCδpal) consists of pSV2neo sequences bounded on one side by homology to the μ gene constant (Cμ) region and on the other side by homology to the δ gene constant (Cδ) region. The Cμ and Cδ flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cμ and Cδ regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cμ and Cδ regions. In several recombinants, palindrome markers in the Cμ and Cδ regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.

ACKNOWLEDGMENTS

This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) (MOP-14416) to M.D.B. and a CIHR Post-Doctoral Fellowship award to J.L.

We thank Steven Raynard, Patricia Bell, and Richard McCulloch, members of our laboratory, for their helpful comments during the course of this work. We thank Erin Wever for excellent technical assistance.