Reliable method of isolating transfected clones from the LNCaP human prostatic cell line.

The LNCaP human prostatic cell line is the only human prostatic cell line available that is both androgen-sensitive and nonmetastatic when implanted into BALB/c/nu/nu mice (4). These properties make it a frequently used model for prostate cancer research. The generation of transfected LNCaP cell lines and the subsequent isolation of transfected clones has been reported by several researchers (3,6,7). Two cloning procedures often used to clone LNCaPs are the cloning ring method (2) and the limit dilution method (5). However, these methods are difficult and tedious and only produce low clone numbers. Two properties of LNCaP cells make them a particularly difficult cell line to clone. First, LNCaP cells grow in loosely adherent colonies (4) and therefore can easily break away from the main colony. This can make cloning rings unreliable. Second, LNCaP cells have a poor survival rate when plated at very low concentrations in tissue culture flask (unpublished observation), which can affect cloning capacity and efficiency when attempting to produce clonal lines using the limit dilution technique. To overcome these difficulties in cloning LNCaP cells, we have developed a simple method for isolating clonal LNCaP cells that have been transfected using cationic liposomemediated transfection (1) with a stable antibiotic resistance marker. We used LNCaP cells (ATCC, Rockville, MD, USA) transfected using LIPOFECTIN Reagent (Life Technologies, Gaithersburg, MD, USA) with the eukaryotic expression vector pRC/CMV (Invitrogen, San Diego, CA, USA), which contains the neomycin-resistance gene for selection. Transfected cells were then selected in 100 μg/mL of antibiotic G418 (GENETICIN; Life Technologies) over 10 days. Because the presence of G-418 was observed to slow down LNCaP cell growth, it was removed, and the heterogeneous population of transfected cells expanded in its absence. These cells have remained stably transfected for at least 60 passages and retained G-418 resistance when reexposed to this antibiotic. Since G-418 resistance was maintained over the period of study, clones could be established from any passage. Transfected LNCaPs at 60%–70% confluence in a 75-cm2 tissue culture flask (Corning Costar, Cambridge, MA, USA) were rinsed with phosphatebuffered saline (PBS). The PBS was removed, and 3 mL 0.2 M EDTA, pH 7.5 were added for 30 s. Following removal of EDTA, cells were incubated in 0.5% trypsin and 5.3 mM EDTA for 15 s, and the excess trypsin was removed. The flask was then placed at 37°C for 5 min; then 3 mL RPMI 1640 medium from the MultiCel Kit (with L-glutamine, without sodium bicarbonate) and 10% fetal calf serum (FCS) from the MultiSer Kit (both kits from Trace Biosciences, Castle Hill, Australia) were added. The LNCaPs were then further diluted in RPMI 1640, 10% FCS to a concentration of 25 cells/mL. Forty microliters of this cell suspension were added to each well in a Linbro 96well plate (ICN Biomedicals, Costa Mesa, CA, USA) so that, theoretically, each well should only contain one cell. The cells were then left overnight to adhere. The next day, the cell number per well was assessed using a phase-contrast microscope. Two hundred microliters of untransfected LNCaP cells at a concentration of 1 × 104 cells/mL were added to wells that were confirmed to contain one cell. The addition of these untransfected cells acts as a feeder layer and removes the need for addition of any exogenous cytokines or hormones. Cellular confluence was monitored daily, and cells were passaged when