Transcriptional silencing of retrovirus vectors in stem cells is a major obstacle to current gene therapy approaches. This phenomenon is influenced by the viral integration site. Insulator elements can overcome this problem by protecting transgenes from position effects. The best studied insulator from the chicken -globin locus (HS4) has to date had limited effectiveness in viral vectors. A novel insulator has been discovered within the D4Z4 repeat sequence that is involved in human Facio-scapulo-humeral dystrophy (FSHD) (Ottaviani, Rival-Gervier et al, PLOS genetic, in press). Protection from silencing is dependent on CTCF and A-type lamin binding at the 5’ end of D4Z4. We evaluate here the effect of D4Z4 on the silencing of retrovirus vectors in mouse embryonic stem (ES) cells. We used the HSC1 self-inactivating retrovirus vector (Osborne et al, 1999. J of virology, 73 (7), p. 5490) containing the eGFP reporter gene controlled by the human EF1gene promoter. This vector was partially insulated by two core HS4 insulators in the LTRs (Yao et al 2003. Nucl. Acid. Res., 31 (18), p. 5317). The D4Z4 insulator fragments were added between gag gene and the EF1 gene promoter. The full length D4Z4 fragment (3.3kb) failed to transmit, but 4 subfragments spanning D4Z4 cloned in both orientations produced titers on NIH 3T3 cells of 105 to 106 IU/ml. Thus D4Z4 subfragments can be transmitted through the virus. Mouse J1 ES cells and NIH3T3 fibroblasts were infected at low MOI to obtain one provirus copy per cell. However, the potential effect of D4Z4 on silencing in ES cells was obscured by the large uninfected cell population. In order to evaluate the effect of D4Z4 on long-term expression, we chose to enrich a population of GFP positive cells by selection. We incorporated an eGFP-puromycin bicistronic gene into our vectors. Infected cells were quickly selected with puromycin and then maintained without further selection. The long-term eGFP expression was followed by flow cytometry. In NIH 3T3 cells, negligible silencing of eGFP expression was detected over 80 days of culture. On the other hand, in infected mouse ES cells, we observed strong silencing of control vectors over 150 days resulting in eGFP+ frequencies ranging from 40% without an insulator to 70% with HS4 in the LTRs. In contrast, the D4Z4 vectors express eGFP reporter in 98% of cells up to 5 months after infection without any selection. A similar sized random sequence failed to protect, suggesting that it is not a spacing effect. Surprisingly, the 3’ subfragment of D4Z4 protects HSC1 provirus from silencing despite lacking the known CTCF and lamin binding sites. This D4Z4 fragment may function through a different mechanism, through novel binding sites, or by cooperating with HS4 elements in the LTR. In order to characterize long-term silencing and protection by D4Z4, DNA methylation and chromatin modification patterns are being characterized. Our results indicate that the D4Z4 insulator maintains stable expression of the HSC1 vector in stem cells over time, and is well suited for stem cell gene therapy applications.