Chesnut J D, Baytan A R, Russell M, Chang M P, Bernard A, Maxwell I H, Hoeffler J P
Invitrogen Corporation, San Diego, CA 92121, USA.
J Immunol Methods. 1996 Jun 14;193(1):17-27. doi: 10.1016/0022-1759(96)00032-4.
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 pCR31acZ (expresses beta-galactosidase), selected, and assayed for beta-galactosidase activity. The positive correlation between sFv and beta-galactosidase expression in these cells (95% of selected cells also expressed beta-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 homogenous 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 beta-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.
我们在此展示一种用于从培养的总细胞群体中快速筛选瞬时转染细胞的新技术。该系统利用重组抗体技术，通过在转染细胞表面展示半抗原结合单链抗体（sFv）来产生一个“分子钩”。来自多个来源的哺乳动物细胞系用编码与跨膜锚定融合的sFv的质粒（pHook-1）进行瞬时转染，结果发现它们在细胞膜上表达并展示功能性半抗原结合sFv。转染细胞凭借其与包被半抗原的磁珠结合的能力从培养的总细胞群体中被筛选出来。一些细胞系在转染后2小时就能够展示足以用于筛选的sFv。SK-BR-3人乳腺癌细胞与pHook-1和pCR31acZ（表达β-半乳糖苷酶）共转染，进行筛选，并检测β-半乳糖苷酶活性。这些细胞中sFv与β-半乳糖苷酶表达之间的正相关关系（95%的筛选细胞也表达β-半乳糖苷酶活性）表明，pHook-1将有助于分离共表达感兴趣的外源基因的细胞。构建了另一种载体，其中感兴趣的基因可以与sFv“钩”从同一质粒表达。这种构建体（pHook-2）允许在不进行共转染或产生稳定转染体的情况下筛选表达外源基因的同质细胞群体。在从该单体质粒中与sFv“钩”共表达lacZ基因的实验中，用抗原包被的磁珠筛选的293人肾细胞和SK-BR-3细胞中有100%对β-半乳糖苷酶活性呈阳性染色。我们认为该系统将成为研究各种野生型和突变蛋白表达的急性和慢性影响的有价值工具。
