Development of a high-efficiency method for gene marking of Dunning prostate cancer cell lines with the enzyme β-galactosidase
✍ Scribed by Rinker-Schaeffer, Carrie W.; Wharam, James F.; Simons, Jonathan; Isaacs, John T.
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 416 KB
- Volume
- 29
- Category
- Article
- ISSN
- 0270-4137
No coin nor oath required. For personal study only.
✦ Synopsis
Although the bacterial enzyme P-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 P-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 P-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 P-galadosidase substrate. Using a modification of standard staining procedures, small metastatic foci of cells expressing P-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 P-galactosidase gene and the stability of its expression in a variety of Dunning sublines.