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Molecular and Cellular Biology, January 2001, p. 501-510, Vol. 21, No. 2
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.2.501-510.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Julang Li,1,* Leah R. Read,2 and Mark D. Baker1,2

Department of Molecular Biology and Genetics1 and Department of Pathobiology,2 University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received 14 July 2000/Returned for modification 25 August 2000/Accepted 18 October 2000

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-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCµCdelta pal) 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 delta  gene constant (Cdelta ) region. The Cµ and Cdelta 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 Cdelta 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 Cdelta regions. In several recombinants, palindrome markers in the Cµ and Cdelta 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.

* Corresponding author. Present address: Department of Animal and Poultry Science, Ontario Agricultural College, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 2713. E-mail: jli{at}uoguelph.ca.

Molecular and Cellular Biology, January 2001, p. 501-510, Vol. 21, No. 2
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.2.501-510.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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