Fig. 1

Restriction of HIV-1-based vectors in murine iPSC. a Schematic overview of integrated LV and GV used in this study. Vectors contain two LTR with deleted U3 regions (∆U3, self-inactivating design), flanking an EGFP expression cassette driven by an EFS enhancer/promoter. SD splice donor, Ψ retroviral packaging signal, RRE rev responsive element, SA splice acceptor, cPPT central polypurine tract, wPRE woodchuck hepatitis virus posttranscriptional regulatory element. b Scheme of reprogramming murine fibroblasts into iPSC by retroviral expression of Oct4, Sox2, Klf4 and c-Myc transcription factors. c iPSC transduced with three independently produced viral supernatants of LV and GV at an MOI of 10 and 100 (n = 3). Flow cytometry data of collected EGFP and SSEA1 (pluripotency marker) double positive cells are shown. NTD non-transduced control. One-way ANOVA with Tukey-Kramer post hoc test was used for statistical analyses. ns (not significant) p = 0.258; *** p ≤ 0.001 (left panel). LV (n = 6) or GV (n = 4) applied to iPSC at an MOI of 100. Mean vector copy numbers per cell were determined 6–8 days after transduction with SYBR Green-based quantitative real-time PCR, based on EGFP copies, and normalized to endogenous PTBP2 DNA copies. The unpaired t test with Welch’s correction was used for statistical analysis. * p = 0.017 (right panel). d LV and GV were applied to different iPSC clones at an MOI of 100. The percentage of EGFP and SSEA1 double positive cells was determined by flow cytometry. One-way ANOVA with Tukey-Kramer post hoc test was used for statistical analyses. #1 ** p = 0.004; #2, #2EX, #3 and #4 *** p ≤ 0.001. e LV and GV from three independently produced supernatants were applied to ESC at an MOI of 10 and 100. Analyses were performed 6–8 days after transduction. The percentage of EGFP and SSEA1 double positive cells was measured by flow cytometry. One-way ANOVA with Tukey-Kramer post hoc test was used for statistical analyses. ns p = 0.660; *** p ≤ 0.001 (left panel). Mean vector copy numbers per cell were determined with SYBR Green-based quantitative real-time PCR, based on EGFP copies, and normalized to endogenous PTBP2 DNA copies and a plasmid standard (right panel)