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Nucleic Acids Research, 1992, Vol. 20, No. 12 2971-2976
© 1992

MOLECULAR BIOLOGY

Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells

J.H. Riley, J.E.N. Morten and R. Anand

ICI Pharmaceuticals, Biotechnology Department Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

Received April 10, 1992. Revised May 15, 1992. Accepted May 15, 1992.

Vectors have been constructed for the introduction of the neomycin resistance gene (neo) Into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the {alpha}-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC.


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