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Publication type: Journal Article
Document type: Full Paper

Year: 2006

Author(s): Hofmann, A; Kessler, B; Ewerling, S; Kabermann, A; Brem, G; Wolf, E; Pfeifer, A

Title: Epigenetic regulation of lentiviral transgene vectors in a large animal model.

Source: Mol Ther. 2006; 13(1):59-66



Authors Vetmeduni Vienna:

Brem Gottfried

Vetmed Research Units
Institute of Animal Breeding and Genetics


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.

Keywords Pubmed: Animals
Animals, Genetically Modified*/metabolism
Azacitidine/pharmacology
Cells, Cultured
CpG Islands
DNA Methylation
DNA Modification Methylases/antagonists & inhibitors
DNA Modification Methylases/metabolism
Epigenesis, Genetic*
Fibroblasts/drug effects
Fibroblasts/metabolism
Genetic Vectors
Green Fluorescent Proteins/genetics
Green Fluorescent Proteins/metabolism
HIV-1/genetics*
Histone Deacetylase Inhibitors
Histone Deacetylases/metabolism
Hydroxamic Acids/pharmacology
Promoter Regions, Genetic
Proviruses/genetics
Swine/genetics*
Swine/metabolism


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