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 reporter gene in a variety of mammalian systems; the variability and instability of its expression has limited its use. Transfection of Dunning 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 defective retrovirus containing a beta-galactosidase expression cassette. Between 30-50% of cells transduced expressed high levels of this enzyme. Homogeneous cell populations were isolated by subsequent fluorescence-activated cell sorting, using a fluorescent beta-galactosidase substrate. Using a modification of standard staining procedures, small metastatic foci of cells expressing beta-galactosidase in mouse lung tissue were detected with high sensitivity. This method has several advantages over standard transfection protocols, including the expedient and efficient transfer of the beta-galactosidase gene and the stability of its expression in a variety of Dunning sublines.

Duke Scholars

Author J. Franklin Wharam Medicine, General Internal Medicine