SELECTIVE ISOLATION OF TRANSIENTLY TRANSFECTED CELLS FROM A MAMMALIAN-CELL POPULATION WITH VECTORS EXPRESSING A MEMBRANE-ANCHORED SINGLE-CHAIN ANTIBODY

We present here a novel technology for the rapid selection of transien tly transfected cells from total populations in culture. This system u tilizes recombinant antibody technology to produce a 'molecular hook' by displaying a hapten-binding single-chain antibody (sFv) on the surf ace of transfected cells. Mammalian cell lines fi om several origins w ere transiently transfected with a plasmid (pHook-1) that encodes an s Fv fused with a transmembrane anchor and found to express and display the functional hapten-bindine sFv on their membranes. Transfected cell s were selected from total populations in culture by virtue of their a bility to bind to hapten-coated magnetic beads. Some cell lines were a ble to display sFv sufficient for selection as early as 2 h post-trans fection. SK-BR-3 human breast carcinoma cells were co-transfected with pHook-1 and pCR3lacZ (expresses beta-galactosidase), selected, and as sayed for beta-galactosidase activity. The positive correlation betwee n sFv and beta-galactosidase expression in these cells (95% of selecte d cells also expressed beta-galactosidase activity) suggests that pHoo k-1 will be useful in isolating cells co-expressing an exogenous gene of interest. Another vector was constructed in which a gene of interes t may be expressed from the same plasmid as the sFv 'hook'. This const ruct (pHook-2) allows the selection of a homogenous population of cell s expressing exogenous genes without co-transfection or the generation of stable transfectants. In experiments where the lacZ gene was co-ex pressed with the sFv 'hook' from this single plasmid, 100% of 293 huma n kidney cells and 100% of SK-BR-3 cells selected with antigen-coated magnetic beads stained positively for beta-galactosidase activity. We propose that this system will be a valuable tool for studying the acut e and chronic effects of the expression of a variety of wild type and mutant proteins.