DEVELOPMENT OF A HIGH-EFFICIENCY METHOD FOR GENE MARKING OF DUNNING PROSTATE-CANCER CELL-LINES WITH THE ENZYME BETA-GALACTOSIDASE

Although the bacterial enzyme beta-galactosidase has been used as a re porter gene in a variety of mammalian systems, the variability and ins tability of its expression has limited its use. Transfection of Dunnin g rat prostatic cell lines with beta-galactosidase expression plasmids resulted in 5-10% of cells expressing the enzyme transiently, and <5% of G418-resistant clones showing any level of expression. To address this problem, we developed a labeling protocol using a replication def ective retrovirus containing a beta-galactosidase expression cassette. Between 30-50% of cells transduced expressed high levels of this enzy me. Homogeneous cell populations were isolated by subsequent fluoresce nce activated cell sorting, using a fluorescent beta-galactosidase sub strate. Using a modification of standard staining procedures, small me tastatic foci of cells expressing beta-galactosidase in mouse lung tis sue were detected with high sensitivity. This method has several advan tages over standard transfection protocols, including the expedient an d efficient transfer of the beta-galactosidase gene and the stability of its expression in a variety of Dunning sublines. (C) 1996 Wiley-Lis s, Inc.