title: 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 γ-retroviral (γ-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.
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Friederike
authors.initials: F
authors.lastname: Herbst
authors.forename: Claudia R
authors.initials: CR
authors.lastname: Ball
authors.forename: Francesca
authors.initials: F
authors.lastname: Tuorto
authors.forename: Ali
authors.initials: A
authors.lastname: Nowrouzi
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Oksana
authors.initials: O
authors.lastname: Zavidij
authors.forename: Sebastian M
authors.initials: SM
authors.lastname: Dieter
authors.forename: Sylvia
authors.initials: S
authors.lastname: Fessler
authors.forename: Franciscus
authors.initials: F
authors.lastname: van der Hoeven
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kloz
authors.forename: Frank
authors.initials: F
authors.lastname: Lyko
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D002454:Cell Differentiation; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D018703:Founder Effect; D020868:Gene Silencing; D020043:Genes, Essential; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D013155:Spleen Focus-Forming Viruses / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D019076:Transgenes
chemical_list: D002352:Carrier Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins