Transfer of P1 inserts into a yeast-bacteria shuttle vector by co-transformation mediated homologous recombination.
Manipulation of genomic inserts cloned into the bacteriophage P1 vector is hindered by the large size of the inserts. We have used co-transformation mediated recombination between the yeast-bacteria shuttle vector, pClasper, and various P1 clones to transfer the entire insert from the P1 into pClasper. This results in the insert being stably maintained in yeast, facilitating mutagenesis by homologous recombination. The recombinant plasmid can subsequently be transferred to and stably maintained in bacteria for efficient plasmid preparation. This method can also be applied to inserts from P1 artificial chromosome or bacterial artificial chromosome vectors.
Criswell
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Department of Biological Sciences, PO Box 210006, University of Cincinnati, Cincinnati, OH 45221-0006, USA.
Bradshaw
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D017101:Bacteriophage P1 / Q000235:genetics*; D002472:Cell Transformation, Viral; D004926:Escherichia coli / Q000378:metabolism*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism*