Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo

Abstract

Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in-retroviral (-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…Transcription elongation NSD1, NSD2 [263] , SET2 [264] , SMYD2 [232] , MMSET [265] ASH1 [266] , JHDM1 [267] , JHDM1A/KDM2A, JHDM1B/KDM2B [268] ISW1B [269] H4K20 me1 me2 me3 (non-genic regions, centromeric heterochromatin, satellite sequences, long terminal repeats Transcritional silencing, heterochromatin, repression of proinflammatory genes PR-SET7/SET8 [270] SUV420H1, SUV420H2 [274] SUV420H2 [274] , SMYD5 [275] PHF8 [271] PHF2 [275] PHF2 [275] L3MBTL1 [272] PHF20 [276] , L3MBTL1 [277] NcoR [275] [ gene [158] . Furthermore, it is well known that a considerable amount of integrated vectors become silent [159] , and this effect seems to be dependent on the promoter chosen to drive the ectopic expression of the gene [160,161] .…”

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

“…However, the implementation of such strategies has been slowed down by the silencing of transgenic promoter/enhancer elements in iPSC/ESCs and their differentiated progeny [3][4][5][6][7][8]. This problem has become particularly obvious for transgenes expressed from c-retroviral or lentiviral vector constructs, which may be silenced by a number of epigenetic mechanisms such as DNA methylation or histone modification [9,10].…”

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

“…We expected that the 914-bp promoter in the transgene could be highly methylated, as is the case for other transgenes (Herbst et al, 2012;Mehta et al, 2009;Pedram et al, 2006). Indeed, we detected a high level of DNA methylation in one line whose transgene was silenced (Fig.…”