Silencing kinetics and reactivation of ERV reporters integrated in a specific genomic site. (A) Scheme for targeting of ERV reporters into a specific genomic site in mESCs via recombinase-mediated cassette exchange (RMCE). The mESC line HA36 contains a hygromycin B and herpes simplex virus thymidine kinase (HyTK) cassette between inverted Lox sites (L1 and 1L). MFG-green fluorescent protein (GFP), MusD-GFP and IAP-GFP proviral reporter cassettes, which contain the Moloney murine leukaemia virus, MusD (approximately +130 bp of downstream sequence) and IAP (approximately +450 bp of downstream sequence) LTRs, respectively, flanked by L1 and 1L sites, were cotransfected into the HA36 line with a Cre recombinase expression vector. Negative selection with ganciclovir eliminated cells with the original HyTK cassette, yielding pools of cells harbouring the proviral reporter cassettes predominantly integrated at the same site. (B) The kinetics of silencing of the MFG, MusD and IAP cassettes after reactivation of the RMCE pool with siRNA against Setdb1 are shown. (C) GFP-negative cells were sorted at day 12 postinduction with Setdb1 siRNA. Robust reactivation of GFP } expression from each of these pools of cells was observed upon secondary Setdb1 knockdown (KD). Flow cytometry data are presented as contour plots and histograms of 10,000 viable (propidium iodide (PI)-negative) cells.