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Nucleic Acids Research, 1993, Vol. 21, No. 7 1541-1548
© 1993

MOLECULAR BIOLOGY

The effects of terminal heterologies on gene targeting by insertion vectors in embryonic stem cells

Satish Kumar and J.Paul Simons*

Department of Molecular Genetics, AFRC Institute of Animal Physiology and Genetics Research Edinburgh Research Station, Roslin, Midlothian EH25 9PS, UK

* To whom correspondence should be addressed

Received January 29, 1993. Revised March 3, 1993. Accepted March 3, 1993.

We have examined the effects of placing non-homologous DNA on the ends of an insertion-type gene targeting vector. The presence of terminal heterologies was found to be compatible with insertion targeting, and the terminal heterologies were efficiently removed. Terminal heterologies reduced the frequency of gene targeting to variable extents. The degree of inhibition of targeting was dependent on the length and the position of the heterology: 2.1kb heterologous sequences were more inhibitory than shorter regions of heterology, and heterology placed on the end of the long (4.8kb) arm of homology was more inhibitory than heterology positioned on the end of the short (0.8kb) arm. When heterology was placed on both arms of the targeting vector the targeting efficiencies were similar to or higher than when heterology was present on the long arm only. These results suggest that terminal sequences are removed simultaneously from both ends of targeting vectors. The removal of terminal sequences probably occurs by exonucleolytic degradation of both strands at each end, and removal of at least one of the strands is intimately coupled with the process of homologous recombination. These findings have implications for the design of gene targeting vectors.


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