Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells.

摘要: A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing cloned Escherichia coli gene asparagine synthetase (AS) into pBR322-simian virus 40 recombinant vector pSV2 so as to place translational initiator codon bacterial AS about 1,000 base pairs downstream from simian early promoter. This construct, pSV2-AS, retains sequences transcriptional and initiation can express bacteria. The construct also complement AS- mutants mammalian cells, giving AS+ transfectants capable growth medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram DNA 10(6) cells exposed). be amplified up 100-fold such selection asparagine-free increasing concentrations inhibitor beta-aspartyl hydroxamate. were found much more resistant second inhibitor, Albizziin, than normal animal lines. difference, which may indicate strong resistance enzyme exploited develop an effective number wild-type lines rat, Chinese hamster, mouse, human origin. LR-73 hamster line, transfected pSV2-AS 0.5 cells. integrated these incubation Advantages disadvantages this dominant, selectable, marker over markers commonly used are discussed.