Membrane-bound neomycin phosphotransferase confers drug-resistance in mammalian cells: a marker for high-efficiency targeting of genes encoding secreted and cell-surface proteins.
An efficient method for inactivating genes is the use of silent selectable markers that are expressed only after homologous recombination into the active target gene. However, use of this approach for genes encoding secreted or membrane-anchored proteins may produce hybrid proteins comprising the N-terminal signal sequence from the target gene linked to the protein conferring drug resistance. Such chimeric enzymes will be secreted, precluding selection for drug resistance. To overcome this problem, we tested the possibility of anchoring in the membrane the cytoplasmic neomycin phosphotransferase (NPT). We constructed a fusion gene with a transmembrane domain connecting the N-terminal signal sequence of a membrane-targeted protein and the neo gene. Expression of this gene yielded G418-resistant colonies of C2C12 cells which contained assayable NPT activity. Comparison of enzyme activity in cell extract fractions verified that the active fusion protein was insoluble, presumably through localization to a membrane compartment. Transmembrane neo cassettes should serve as integration-activated markers capable of targeting genes encoding secreted or cell surface proteins.
Mohler
W A
WA
Department of Molecular Pharmacology, Stanford University, California 94305-5332.
Blau
H M
HM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015415:Biomarkers; D002478:Cells, Cultured; D017931:DNA Primers; D004351:Drug Resistance; D018390:Gene Targeting; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology*; D019868:Kanamycin Kinase; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D017931:DNA Primers; D005839:Gentamicins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C010680:antibiotic G 418; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase