Gene Transfer and Expression Protocols

Bujdoso, R, Sargan, D, Ballingall, K, Sanderson, A, Raymond Bujdoso, David Sargan, Keith Ballingall, Andrew Sanderson, Bujdoso, Raymond, Sargan, David, Ballingall, Keith, Sanderson, Andrew

Flow cytometry and fluorescence-activated cell sorting (FACS) are techniques of great power used to screen cells rapidly for expression of particular gene products. These techniques have been of general utility in identifying and selecting populations of cells of defined characteristics from body fluids and other natural sources, More recently they have received extensive attention as methods for screening cell-surface expressed gene products in transfected cells. These methods rely on the indirect coupling of detector molecules, usually fluorochromes, to specific molecules on the target cells. This may occur through conjugation of the fluorochrome to the ligand of a receptor, or, as is more generally the case, through the use of fluorochromeconjugated antibodies specific for the transfected gene product. Cells displaying specific surface fluorescence following exposure to a flurochrome conjugate may subsequently be positively selected (or excluded) by FACS. Since cells are sorted individually, FACS is an ideal technique for picking up very rare events and for finding very minor subpopulations. In theory at least, the experimenter may recover a single cell of the desired phenotype from a relatively large population. However, the examination of single cells.