Selection of Transfected Cells

Abstract

DNA-mediated gene transfer techniques have revolutionalized molecular biology and are used extensively to study the function and regulation of eukaryotic genes in a variety of cell types. In general, expression of genes in mammalian cells can be studied by either stable transformation or by transient expression. Stable transformation of cells expressing the gene of interest can be achieved by cotransfection of the gene cloned in an appropriate expression vector under the control of a constitutive or inducible eukaryotic cellular or viral promoter and a vector that carries a dominant selectable marker such as Eco-gpt (1) or neo (2). Since the DNA-mediated gene transfer methods target only a fraction of cells for gene expression, the isolation of stably transfected cells with a selectable marker gives rise to a cell population expressing the gene of interest free of untransfected cells under conditions of drug selection. However, this method of selection is a long-term process and may have adverse effects on host cell functions such as cell growth or chromosomal rearrangements due to integration of a single or multiple copies of the selectable marker gene. Transient expression without a dominant selectable marker allows functional analysis of the transfected gene within 24–72 h after transfection but suffers from the drawback that the presence of a large fraction of untransfected cells in the milieu of cells expressing the gene of interest may give rise to problems of interference due to high background. Therefore, it was necessary to develop a method for isolation of transiently transfected cells free of untransfected cells within 24–72 h after transfection. With this overall goal in mind, the previously developed “panning” methodology was modified to isolate transiently transfected cells expressing the gene of interest together with a cotransfected cell surface marker gene using the magnetic affinity cell sorting (MACS) technology (3; for reviews, see refs 4–8). The MACS methodology allows the separation of cells expressing a surface protein away from those lacking the marker. The cell surface marker could be either introduced into cells by DNA-mediated gene transfer techniques or be an endogenously-expressing protein on the surface of selective cell type. In either case, the antibodies against the surface protein attached to a magnetic matrix are used to selectively “pull out” cells expressing that surface marker with the application of a magnetic field under appropriate experimental conditions whereas the cells lacking the marker remain unaffected. Expression of any cell surface protein for which a suitable antibody is available in a variety of cell types using DNA-mediated gene transfer methods allows this methodology to be useful in a wide range of biological applications (6,8,9). In the initial stages of development of this application of MACS methodology to transfected cells, readily assayable reporter gene product such as chloramphenicol acetyltransferase (CAT) was used as the gene of interest in conjunction with the cell surface markers such as the vesicular stomatitis virus glycoprotein (VSV-G) and the Tat subunit of interleukin 2 receptor (IL-2R) for transient expression in mammalian cells by DNA-mediated transfection techniques (3,10,11). Subsequently, it was shown that MACS could be successfully used to select a rare population of cells expressing the P-glycoprotein, the product of multiple drug resistant (mdr) gene (6,8,9) among human lymphomas as well as for selection of virus-infected cells expressing a surface protein (using dengue virus as an example) (6). The experimental conditions for MACS methodology have undergone some improvement over the original protocol published (3), and the modified procedure is described in this chapter.