Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse.
We constructed a series of cosmid vectors that carry two tandemly arranged lambda cos and mammalian selective markers. We achieved cloning efficiencies of 1-3 x 10(7) and greater than 10(6) colony-forming units per microgram of insert, using a cloned 42-kb BamHI fragment and Sau3AI fragments of 40-50 kb from mouse genomic DNA, respectively. The modified Ca.phosphate coprecipitation method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616] considerably improved the efficiency of gene transfer of cosmids into cultured mammalian cells: when genes encoding thymidine kinase from herpes simplex virus type 1 and aminoglycoside 3'-phosphoribosyltransferase from Tn5 were selected, the efficiencies of gene transfer into mouse L cells were about 10(-6). The mouse genome contains one copy of the functional gene for elongation factor 2 (EF2) per haploid genome and multiple copies of the EF2-related gene. We isolated a cosmid that carried functional full-length mouse EF2 from a cosmid library of L-cell genomic DNA, by colony hybridization and subsequent gene transfer of candidate cosmids into human 143B cells.[1]References
- Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse. Ishiura, M., Ohashi, H., Hazumi, N., Uchida, T., Okada, Y. Gene (1989) [Pubmed]
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