Although the bacterial enzyme β-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 β-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 β-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 β-galactosidase substrate. Using a modification of standard staining procedures, small metastatic foci of cells expressing β- 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 β-galactosidase gene and the stability of its expression in a variety of Dunning sublines.
|Original language||English (US)|
|Number of pages||5|
|State||Published - Jul 1996|
- β-galactosidase tagging method
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