Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane anchored single-chain antibody
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- 作者：Chesnut ; Jonathan D. ; Baytan ; Apollo R. ; Russell ; Marijane ; Mei-Ping ; Chang ; Bernard ; Amy ; et. al.
- 关键词：Transient transfection ; Single-chain antibody ; Eukaryotic expression vector ; Membrane display ; Gene expression
- 刊名：Journal of Immunological Methods
- 出版年：1996
- 出版时间：June 14, 1996
- 年：1996
- 卷：193
- 期：1
- 页码：17-27
- 全文大小：1.16 M
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 produce a ‘molecular hook’ by displaying 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. Transfected cells were selected from total populations in 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 pCR3lacZ (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 also expressed β-galactosidase activity) suggests that pHook-1 will be useful in isolating cells co-expressing an exogenous gene of interest. Another vector was constructed in which a gene of interest may be expressed from the same plasmid as the sFv ‘hook’. This construct (pHook-2) allows the selection of a homogeneous population 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 single 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 studying the acute and chronic effects of the expression of a variety of wild type and mutant proteins.