Replica Filter Screening Technique to Detect Transfected Cells Expressing β 2 -Adrenergic Receptor

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.