The mechanism of mammalian gene replacement is consistent with the formation of long regions of heteroduplex DNA associated with two crossing-over events.
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 mu-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCmuCdeltapal) consists of pSV2neo sequences bounded on one side by homology to the mu gene constant (Cmu) region and on the other side by homology to the delta gene constant (Cdelta) region. The Cmu 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 Cmu 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 Cmu and Cdelta regions. In several recombinants, palindrome markers in the Cmu 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.
Li
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J
Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada N1G 2W1. jli@uoguelph.ca
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D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics; D002460:Cell Line; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D003434:Crossing Over, Genetic / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004274:DNA, Recombinant / Q000032:analysis / Q000235:genetics; D005785:Gene Conversion / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006825:Hybridomas / Q000166:cytology / Q000378:metabolism; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007138:Immunoglobulin delta-Chains / Q000235:genetics; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D051379:Mice; D008957:Models, Genetic; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004274:DNA, Recombinant; D005819:Genetic Markers; D007127:Immunoglobulin Constant Regions; D007138:Immunoglobulin delta-Chains; D007148:Immunoglobulin mu-Chains; D009692:Nucleic Acid Heteroduplexes