A severe de novo methylation of episomal vectors by human ES cells.
Episomal vectors can allow efficient genetic modification of cells and have the potential advantage of avoiding chromosomal position of integration effects. Here we explore the use of an Epstein-Barr virus-based episomal vector with human embryonic stem (ES) cells, and find high initial transfection rates, but a rapid loss of reporter gene expression. Similar to mouse ES cells, human ES cells express high levels of the de novo DNA methyltransferases, and we detected dramatic CpG methylation and minor non-CpG methylation on the episomes recovered from the human ES cells 7 days after the transfection, which was not present on the same episome recovered from 293 cells. Interestingly, the oriP region of the episomes was relatively excluded from this methylation. These findings define some of the limitations of using episomal vectors with human ES cells and offer a unique platform for analyzing epigenetic gene silencing in human ES cells.
Kameda
Takashi
T
The Genome Center of Wisconsin, University of Wisconsin-Madison, 53706, USA.
Smuga-Otto
Kim
K
Thomson
James A
JA
D002478:Cells, Cultured; D019175:DNA Methylation; D005822:Genetic Vectors / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology*; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins