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.
Rinker-Schaeffer
C W
CW
Department of Oncology, Brady Urological Institute, Johns Hopkins School of Medicine, Baltimore, Maryland 21231, USA.
Wharam
J F
JF
Simons
J
J
Isaacs
J T
JT
D000818:Animals; D005434:Flow Cytometry; D005819:Genetic Markers; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D016513:Mice, SCID; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D010957:Plasmids; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics*; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics*
D005819:Genetic Markers; D001616:beta-Galactosidase