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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: J Virol Methods. 2011 Mar 17;174(1-2):12–21. doi: 10.1016/j.jviromet.2011.03.012

Figure 1. Generation of RTA-inducible SLK cells (iSLK.219) harboring recombinant KSHV (rKSHV.219).

Figure 1

A) SLK cells were first transduced sequentially with retroviruses expressing rtTA or RTA, resulting in RTA-inducible SLK (iSLK) cells. iSLK cells were infected with rKSHV.219 and subsequently selected in the presence of 1 µ/ml puromycin, generating iSLK.219 cells. Concentration of puromycin was gradually increased up to 10 µ/ml to increase the copy number of KSHV episome. B) GFP intensity of iSLK.219 cells, maintained at 1 µ/ml and 10 µ/ml, as indicated, is plotted. Gray histograms represent intermediate iSLK.219 cells, selected at 2 µ/ml through 9 µ/ml puromycin. Filled histogram represents uninfected iSLK cells. Note that there is a significant increase in GFP− cells among iSLK.219 cells maintained at 8, 9, and especially at 10 µ/ml puromycin. Note that GFP intensity increases when puromycin concentration increases to maintain iSLK.219 cells. PURO represents puromycin. C) iSLK.219 cells were induced with doxycycline (1 µ/ml) for 2 days and GFP intensity vs % GFP+RFP+ is plotted. Note that GFP+ cells were gated on FACS and mean GFP intensity was calculated. D) Infectious units (GFP-transducing untis) in the induced culture supernatants (d3) of iSLK.219 cells, selected at increasing concentrations of puromycin to boost numbers of episomes in the cells, were measured on QBI293A cells. See Materials and Methods for details.