<p>Generation of stable cell lines</p><p>In this type of experiments you’re selecting for stable expression of a gene, because it’s integrated into the host chromosome. Delivery with retrovirus is generally most efficient.</p><p>1. Cells are initially either transfected or infected with retro or lentivirus. 2. At 48 hrs after gene delivery, selection is started. One can use either of several different selection markers or a combination of these (Puromycin generally 1.5-3 µg/ml, but CV1-P 8 µg/ml, G418 500 µg/ml, hygromycin 200 µg/ml, blasticidin 5 µg/ml). Puromycin usually kills cells in 3 days, G418 in 7 days, hygromycin in 7 days and blasticidin in 5 days. 3. Cells are often split before selection (e.g. 1:3). That’s because selection does not work if cells are too confluent! Cells should not be too sparse either; they would have a difficult time recovering. It’s hard to predict when and how much to split the cells, because it really depends on titer of the virus (how many viral particles/ml) and whether the gene of interest affects cell growth. 4. Cells are usually analyzed for expression and frozen down in aliquots for later use at 7-10 days after transfection/ infection. Maintain cells under selection at all times.</p><p>HM136 mesothelial cells: 30 µg/ml hygromycin! 293H: 1 mg/ml G418</p>