Replica Filter Screening Technique to Detect Transfected Cells Expressing β2-Adrenergic Receptor

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Abstract
We have utilized a replica transfer technique to develop a novel screening assay for the identification of transfectants expressing β2-adrenergic receptors (β2-AR). The hamster β2-AR gene flanked by either its natural promoter or the zinc-inducible mouse metailothionein (MMT) promoter was cotransfected with plasmids conferring neomycin resistance (pRSVneo) into β2-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 β2-AR. Individual colonies were isolated and examined to determine β2-AR gene dosage, mRNA expression, and receptor densities and affinities. Analysis of cell lines expressing β2-AR indicates that this method can identify transfectants containing only a single β2-AR gene copy and expressing as few as 4,000 β2-receptors per cell. This method may be useful as a tool for the molecular cloning of neuro-transmitter receptor genes and for the measurement of transfection efficiencies and expression of receptor genes in cells.