Mechanisms Involved in Targeted Gene Replacement in Mammalian Cells

Corresponding author: Mark D. Baker, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada., mdbaker{at}uoguelph.ca (E-mail)

Communicating editor: L. S. SYMINGTON

The "ends-out" or omega ()-form gene replacement vector is used routinely to perform targeted genome modification in a variety of species and has the potential to be an effective vehicle for gene therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown. Understanding molecular features associated with gene replacement is important and may lead to an increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal µ- locus in a murine hybridoma cell line. The results showed that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.

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				J. Li, L. R. Read, and M. D. Baker The Mechanism of Mammalian Gene Replacement Is Consistent with the Formation of Long Regions of Heteroduplex DNA Associated with Two Crossing-Over Events Mol. Cell. Biol., January 15, 2001; 21(2): 501 - 510. [Abstract] [Full Text] [PDF]