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Nucleic Acids Research, 1995, Vol. 23, No. 14 2784-2790
© 1995

MOLECULAR BIOLOGY

The effect of target site transcription on gene targeting in human cells in vitro

Bhaskar Thyagarajan, Brad L. Johnson and Colin Campbell*

Department of Pharmacology, University of Minnesota Medical School 435 Delaware Street South East,Minneapolis, MN 55455, USA

*To whom correspondence should be addressed

Received February 24, 1995. Accepted June 1, 1995.

We evaluated the effect of target site transcription on gene targeting In cultured human flbrosarcoma cells. A number of cell lines that harbored a plasmid recombination substrate within their chromosomal DNA were created. Gene targeting frequency was then measured at these different loci in the presence and absence of an agent that stimulated target site transcription. We observed that gene targeting was significantly enhanced by RNA transcription. The magnitude of transcription-stimulated gene targeting varied from 3-fold to >20-fold. No increase in gene targeting was observed, however, when transcription proceeded away from, rather than through, the recombination site. Transcription-stimulated gene targeting was also observed when single-stranded plasmid vectors complementary to either the coding or template strand were used as recombination substrates. Our results indicate that gene targeting, like other forms of DNA recombination, can be stimulated by target site transcription. The implications of our observations on current models of transcription-stimulated recombination are discussed.


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