Abstract
The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ΦC31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ΦC31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to ca...Continue Reading
References
Jul 22, 1997·Proceedings of the National Academy of Sciences of the United States of America·D A Siegele, J C Hu
Feb 24, 1998·Gene Therapy·A M DarquetJ Crouzet
Oct 6, 1999·Current Opinion in Biotechnology·F Baneyx
Apr 17, 2001·The Journal of Biological Chemistry·B W BiggerC Coutelle
Aug 30, 2003·Molecular Therapy : the Journal of the American Society of Gene Therapy·Zhi-Ying ChenMark A Kay
Mar 19, 2004·Gene Therapy·Z Y ChenM A Kay
Apr 2, 2004·Nature Reviews. Immunology·Dennis M Klinman
Feb 11, 2005·Human Gene Therapy·Zhi-Ying ChenMark A Kay
Sep 24, 2005·Journal of Molecular Microbiology and Biotechnology·Wolfgang JechlingerPeter Mayrhofer
Feb 7, 2008·Molecular Therapy : the Journal of the American Society of Gene Therapy·Zhi-Ying ChenMark A Kay
Sep 17, 2008·BMC Biology·Georgios TrichasShankar Srinivas
Jan 9, 2009·Gene Therapy·D R GillS C Hyde
Jul 2, 2009·Methods in Molecular Biology·Peter MayrhoferWolfgang Jechlinger
Feb 9, 2010·Nature Methods·Fangjun JiaJoseph C Wu
Nov 26, 2010·Nature Biotechnology·Mark A KayZhi-Ying Chen
Aug 2, 2011·Nature Biotechnology·Xun XuJun Wang
Apr 3, 2012·Molecular Biotechnology·Dennis KobeltWolfgang Walther
May 7, 2013·Journal of Biotechnology·Yixiao DongWenxin Wang