title: Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane anchored single-chain antibody.
abstract: 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.
authors.affiliation: Invitrogen Corporation, San Diego, CA 92121, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Chesnut
authors.forename: A R
authors.initials: AR
authors.lastname: Baytan
authors.forename: M
authors.initials: M
authors.lastname: Russell
authors.forename: M P
authors.initials: MP
authors.lastname: Chang
authors.forename: A
authors.initials: A
authors.lastname: Bernard
authors.forename: I H
authors.initials: IH
authors.lastname: Maxwell
authors.forename: J P
authors.initials: JP
authors.lastname: Hoeffler
mesh_terms: D000595:Amino Acid Sequence; D002469:Cell Separation / Q000379:methods; D002999:Clone Cells; D005822:Genetic Vectors / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics*; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D008969:Molecular Sequence Data; D010081:Oxazolone / Q000276:immunology; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry / Q000235:genetics; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D007128:Immunoglobulin Fragments; D011947:Receptors, Antigen, B-Cell; C055966:immunoglobulin Fv; D010081:Oxazolone; D001616:beta-Galactosidase