Replica filter screening technique to detect transfected cells expressing beta 2-adrenergic receptor.
We have utilized a replica transfer technique to develop a novel screening assay for the identification of transfectants expressing beta 2-adrenergic receptors (beta 2-AR). The hamster beta 2-AR gene flanked by either its natural promoter or the zinc-inducible mouse metallothionein (MMT) promoter was cotransfected with plasmids conferring neomycin resistance (pRSVneo) into beta 2-AR-deficient mouse L cells. Transfectant colonies were grown on polyester nylon filters and screened by filter binding with [125I]iodohydroxybenzylpindolol to identify colonies expressing beta 2-AR. Individual colonies were isolated and examined to determine beta 2-AR gene dosage, mRNA expression, and receptor densities and affinities. Analysis of cell lines expressing beta 2-AR indicates that this method can identify transfectants containing only a single beta 2-AR gene copy and expressing as few as 4,000 beta 2-receptors per cell. This method may be useful as a tool for the molecular cloning of neurotransmitter receptor genes and for the measurement of transfection efficiencies and expression of receptor genes in cells.
Machida
C A
CA
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Bunzow
J
J
Hanneman
E
E
Grandy
D
D
Civelli
O
O
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005796:Genes; D005818:Genetic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011943:Receptors, Adrenergic, beta / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D015345:Oligonucleotide Probes; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D004247:DNA