Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation.
Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection. In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.
Institute of Cell Biology, University of Witten/Herdecke, Witten, Germany.
Andreas C W
AC
Jenke
Monica F
MF
Scinteie
Isa M
IM
Stehle
Hans J
HJ
Lipps
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003587:Cytomegalovirus / Q000235:genetics; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015870:Gene Expression / Q000235:genetics / Q000502:physiology; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D051381:Rats; D020800:Receptor, Nerve Growth Factor / Q000032:analysis / Q000235:genetics; D014162:Transfection; D019076:Transgenes / Q000235:genetics / Q000502:physiology*
D020800:Receptor, Nerve Growth Factor; D003596:Cytosine