Nucleic Acids Research, 1990, Vol. 18, No. 22 6485-6489
© 1990
MOLECULAR BIOLOGY |
High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe
Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University 3-1 Yamadaoka Suita, Osaka 565, Japan
*To whom correspondence should be addressed
Received October 1, 1990. Accepted November 22, 1990.
We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 106 colonies per 108 cells transfected with 2 µg of library and 1 µg of vector, 50–60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz.pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast.
This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.
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