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.
Ishiura
M
M
National Institute for Basic Biology, Aichi, Japan.
Ohashi
H
H
Hazumi
N
N
Uchida
T
T
Okada
Y
Y
D000818:Animals; D003360:Cosmids; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D005796:Genes; D005822:Genetic Vectors; D007739:L Cells / Q000378:metabolism; D051379:Mice; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection
D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D004247:DNA