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.
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