Epigenetic regulation of lentiviral transgene vectors in a large animal model.
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.
Hofmann
Andreas
A
Department of Pharmacy, Molecular Pharmacology, Center for Drug Research, Butenandtstrasse 5 (C), 81377 Munich, Germany.
Kessler
Barbara
B
Ewerling
Sonja
S
Kabermann
Andrea
A
Brem
Gottfried
G
Wolf
Eckhard
E
Pfeifer
Alexander
A
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D001374:Azacitidine / Q000494:pharmacology; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D044127:Epigenesis, Genetic; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics; D013552:Swine / Q000235:genetics* / Q000378:metabolism
D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C012589:trichostatin A; D015254:DNA Modification Methylases; D006655:Histone Deacetylases; D001374:Azacitidine