Construction of a neo fusion gene for expression in both prokaryotic and eukaryotic cells.
A high-copy-number plasmid, pLink, was constructed to allow the direct selection in Escherichia coli of a neo fusion gene capable of conferring Geneticin (G418) resistance on mouse L cells. pLink was derived from pdMmtneo by insertion of a KpnI linker within the 5'-coding region of the neo gene. This created a minus-one frameshift mutation resulting in a translational termination within the N-terminal region of the protein. The Neo activity was restored by insertion into the modified neo gene of a piece of coding sequence derived from human HPRT cDNA. The resulting plasmid, pAH, was microinjected into mouse A9 cells and shown to confer resistance to G418.
AFRC Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, U.K.
A J
AJ
Harwood
A
A
Shervington
C J
CJ
Bostock
D000818:Animals; D001483:Base Sequence; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005839:Gentamicins; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009355:Neomycin; D010443:Peptide Chain Termination, Translational / Q000187:drug effects; D010957:Plasmids; D011993:Recombinant Fusion Proteins; D012641:Selection, Genetic
D005839:Gentamicins; D011993:Recombinant Fusion Proteins; C010680:antibiotic G 418; D007041:Hypoxanthine Phosphoribosyltransferase; D009355:Neomycin