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Nucleic Acids Research, 1992, Vol. 20, No. 11 2693-2698
© 1992

MOLECULAR BIOLOGY

Targeted alterations in yeast artificial chromosomes for inter-species gene transfer

Nicholas P. Davies, Ian R. Rosewell and Marianne Brüggemann*

AFRC, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, UK

*To whom correspondence should be addressed

Received March 23, 1992. Revised May 7, 1992. Accepted May 7, 1992.

In order to facilitate alterations of large DNA molecules for their introduction into mammalian cells we have characterised the mechanism of site-specific modifications in yeast artificial chromosomes (YACs). Newly developed yeast integration vectors with dominant selectable marker genes allow targeted integration into left (centromeric) and right (noncentromeric) YAC arms as well as alterations to the human derived insert DNA. In transformation experiments, integration proceeds exclusively by homologous recombination although yeast prefers linear ends of homology for predefined insertions. Targeted regions can be rescued which expedite the cloning of internal human sequences and the identification of 5' and 3' YAC/insert borders. Integration of the neomycin resistance gene into various parts of the YAC allowed the transfer and stable integration of large DNA molecules into a variety of mammalian cells including embryonic stem cells.


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