| First Author | Ishiura M | Year | 1989 |
| Journal | Gene | Volume | 85 |
| Issue | 2 | Pages | 427-33 |
| PubMed ID | 2628177 | Mgi Jnum | J:10363 |
| Mgi Id | MGI:58815 | Doi | 10.1016/0378-1119(89)90436-8 |
| Citation | Ishiura M, et al. (1989) Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse. Gene 85(2):427-33 |
| abstractText | 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. |
