Escherichia coli gpt as a positive and negative selectable marker in embryonal stem cells.
Transfection of HPRT- L fibroblasts with a plasmid containing two linked selectable markers genes, gpt and neo, regulated by the same eukaryotic control elements, yielded a 6-fold higher transfection frequency on selection for neo than for gpt. Transfection of HPRT- embryonal stem (ES) cells with the same plasmid yielded high levels of transfectants when selected for neo expression with G418, but a level of transfection greater than two orders of magnitude lower was observed when HAT supplemented medium was used to select for gpt expression. Selection for gpt expression in ES cells with medium containing mycophenolic acid and xanthine gave slightly higher frequencies of transfection, but still considerably lower than that for neo selection. In addition, mycophenolic acid exhibited a general cytotoxicity to ES cells with the window between toxicity of this compound to gpt- ES cells and gpt+ ES cells being very narrow. Cells selected with mycophenolic acid and xanthine for expression of gpt remained sensitive to HAT selection. Expression of gpt in a representative ES cell line, selected on mycophenolic acid and xanthine, was verified by Northern analysis and sensitivity to 6-thioguanine. While the level of mRNA expression in this ES cell line was insufficient to support growth via purine salvage when exposed to HAT medium, identical levels of gpt expression in HPRT- L cells, as judged by Northern analysis, allowed for normal growth in HAT medium. This suggests that ES cells place a greater demand on purine nucleotide biosynthesis than L cells. These results are discussed in terms of the use of gpt as a positive and negative selectable marker for gene targeting via homologous recombination in ES cells.
Spring
K J
KJ
Centre for Molecular Biology and Biotechnology, University of Queensland, Brisbane, Australia.
Mattick
J S
JS
Don
R H
RH
D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005821:Genetic Techniques; D005822:Genetic Vectors; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009173:Mycophenolic Acid / Q000494:pharmacology; D010430:Pentosyltransferases / Q000235:genetics*; D011685:Purine Nucleotides / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D013234:Stem Cells / Q000201:enzymology*; D014162:Transfection
D011685:Purine Nucleotides; D012333:RNA, Messenger; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D009173:Mycophenolic Acid