Skip Navigation


Nucleic Acids Research Advance Access originally published online on August 2, 2007
Nucleic Acids Research 2007 35(15):e98; doi:10.1093/nar/gkm570
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (2451K) Freely available
Right arrow Screen PDF (497K) Freely available
Right arrowOA All Versions of this Article:
35/15/e98    most recent
gkm570v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Lufino, M. M.P.
Right arrow Articles by Wade-Martins, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lufino, M. M.P.
Right arrow Articles by Wade-Martins, R.
Related Collections
Right arrow Recombinant DNA expression
Right arrow DNA transfer
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2007, Vol. 35, No. 15 e98
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency

Michele M.P. Lufino1, Roberto Manservigi2 and Richard Wade-Martins1,*

1The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK and 2Department of Experimental and Diagnostic Medicine, Section of Microbiology, University of Ferrara, Via Luigi Borsari, 46, 44100, Ferrara, Italy

*To whom correspondence should be addressed. Tel: +44 (0) 1865 287761; Fax: +44 (0) 1865 287501; Email: richard.wade-martins{at}well.ox.ac.uk or Email: richard.wade-martins{at}dpag.ox.ac.uk

Received May 2, 2007. Revised July 4, 2007. Accepted July 12, 2007.

Episomal gene expression vectors offer a safe and attractive alternative to integrating vectors. Here we describe the development of a high capacity episomal vector system exploiting human episomal retention sequences to provide efficient vector maintenance and regulated gene expression through the delivery of a genomic DNA locus. The iBAC-S/MAR vector is capable of the infectious delivery and retention of large genomic DNA transgenes by exploiting the high transgene capacity of herpes simplex virus type 1 (HSV-1) and the episomal retention properties of the scaffold/matrix attachment region (S/MAR). The iBAC-S/MAR vector was used to deliver and maintain a 135 kb genomic DNA insert carrying the human low density lipoprotein receptor (LDLR) genomic DNA locus at high efficiency in CHO ldlr–/– a7 cells. Long-term studies on CHO ldlr–/– a7 clonal cell lines carrying iBAC-S/MAR-LDLR demonstrated low copy episomal stability of the vector for >100 cell generations without selection. Expression studies demonstrated that iBAC-S/MAR-LDLR completely restored LDLR function in CHO ldlr–/– a7 cells to physiological levels and that this expression can be repressed by ~70% by high sterol levels, recapitulating the same feedback regulation seen at the endogenous LDLR locus. This vector overcomes the major problems of vector integration and unregulated transgene expression.

Present address: Michele M.P. Lufino and Richard Wade-Martins, Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.