Eukaryotic expression vectors for the membrane display of single-chain antibodies: their use in the selection of transiently-transfected cells from total populations in culture.
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文摘
We present here a novel technology for the rapid selection of transiently-transfected cells from total populations in culture. This system utilizes recombinant antibody technology to display a hapten-binding single-chain antibody (sFv) on the surface of transfected cells. Mammalian cell lines from several origins were transiently transfected with a plasmid (pHOOK-1) that encodes an sFv fused with a transmembrane anchor and found to express and display the functional hapten-binding sFv on their membranes. These cells were selected from culture by virtue of their ability to bind to hapten-coated magnetic beads. Some cell lines were able to display sFv sufficient for selection as early as 2 h post-transfection. SK-BR-3 human breast carcinoma cells were co-transfected with pHOOK-1 and pCR31acZ (expresses β-galactosidase), selected, and assayed for β-galactosidase activity. The positive correlation between sFv and β-galactosidase expression in these cells (95 % of selected cells expressed β-galactosidase activity) suggests that pHOOK-1 will be useful in isolating cells expressing an exogenous gene of interest. Another vector has been constructed in which a gene of interest may be expressed from the same plasmid as the sFv ‘hook’. This construct (pHA-NEO) allows the selection of cells expressing exogenous genes without co-transfection or the generation of stable transfectants. In experiments where the lacZ gene was co-expressed with the sFv ‘hook’ from this plasmid, 100 % of 293 human kidney cells and 100 % of SK-BR-3 cells selected with antigen-coated magnetic beads stained positively for β-galactosidase activity. We propose that this system will be a valuable tool for the study the acute and chronic effects of the expression of a variety of wild type and mutant proteins.