Epigenetic Regulation of Lentiviral Transgene Vectors in a Large Animal Model

Abstract

Transgenic animals are of outstanding relevance for genetic studies and the development of novel therapies for human diseases. A recent development is the generation of transgenic animals by lentiviral gene transfer. So far, studies on lentiviral transgenesis focused on first-generation (founder or F0) animals-most of which carry multiple integrants. Here, we analyze transgene expression and epigenetic regulation of individual integrants in lentiviral transgenic pigs after segregation to the F1 generation. Unexpectedly, one-third of lentiviral integrants exhibited low expression levels and were hypermethylated, as demonstrated by methylation-sensitive Southern blotting and bisulfite sequencing. Proviral methylation density correlated inversely with expression levels. In addition, treatment of isolated transgenic fibroblasts with the DNA methylase inhibitor 5-azacytidine induced a threefold increase in mean fluorescence intensity (MFI) from 8 to 26.1. Treatment with the histone deacetylase inhibitor trichostatin A enhanced MFI to only 11.1. Taken together, expression of lentiviral integrants in higher mammals is regulated by epigenetic modifications. In contrast to previous expectations, DNA methylation plays an important role in lentiviral expression.

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…Lentiviral transduction can produce high ratios of transgenic offspring (20-30 % transgenic offspring per treated embryos), but this technique frequently results in cell mosaicism and thus reduced germline transmission; the maximal cargo of foreign DNA with lentiviral vectors is about 7 kbp; and epigenetic silencing of the virally integrated constructs has been observed 13 . Another limitation is that a laboratory with biosafety level 2 is required for lentivirus production and handling.…”

“…Humanized pig models include those for retinitis pigmentosa 6 , cystic fibrosis 7 , Alzheimer´s disease 8 , Huntington´s disease 9 , familial adenomatous polyposis 10 , and immunodeficiency 11 . However, transgenesis in the pig, most commonly achieved by pronuclear DNA injection (PNI) or by somatic cell nuclear transfer (SCNT), is an inefficient and expensive process, hampered by poor predictability of levels and patterns of transgene expression 2,3,[12][13][14] . Gene targeting by homologous recombination is extremely inefficient in porcine somatic cells 3 , and porcine embryonic stem cells (ES) have not yet been established 15 .…”

Genotypic Features of Lentivirus Transgenic Mice

“…Their generation through the injection of naked plasmid DNA into the male pronucleus of fertilized oocytes has been a standard practice for almost 3 decades (5), but the success of this procedure has been largely limited to mice. Recently, lentiviral vector-mediated transgenesis has emerged as an attractive alternative, as these human immunodeficiency virus (HIV)-derived gene transfer vehicles can mediate the efficient integration of their cargo into zygotes or early progenitors from a wide variety of species including mice, rats, pigs, cows, and chickens (4,8,13,20,23). In a typical procedure, the lentivirus particle is injected beneath the zona pellucida of a fertilized oocyte and penetrates this cell by fusion between the viral and plasma membranes.…”

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

“…correlated with increases in H3 histone deacetylation and CpG dinucleotide methylation within the proviral region [15]. Furthermore, epigenetic regulation has also been able to silence lentiviral vector-mediated transgene expression during stem cell differentiation in vivo, in transgenic mice [25,26]. This could limit the efficacy of genetically engineered ASCs for tissue repair/regeneration applications.…”