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.