Skip Navigation


Nucleic Acids Research Advance Access originally published online on June 15, 2009
Nucleic Acids Research 2009 37(17):e111; doi:10.1093/nar/gkp511
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (537K) Freely available
Right arrow Screen PDF (291K) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
37/17/e111    most recent
gkp511v1
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 Wang, S.
Right arrow Articles by Zhu, J.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, S.
Right arrow Articles by Zhu, J.
Related Collections
Right arrow Recombinant DNA expression
Right arrow Recombination
Right arrow Genomics
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2009, Vol. 37, No. 17 e111
© 2009 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

Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome

Shuwen Wang, Yuanjun Zhao, Melanie A. Leiby and Jiyue Zhu*

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA

*To whom correspondence should be addressed. Tel: +1 717 531 3597; Fax: +1 717 531 7667; Email: joz1{at}psu.edu

Received March 23, 2009. Revised May 14, 2009. Accepted May 28, 2009.

The endogenous human telomerase reverse transcriptase (hTERT) gene is repressed in somatic cells. To study the mechanisms of its repression, we developed a strategy of retrovirus-directed Cre recombinase-mediated BAC targeting, or RMBT, to generate single-copy integrations of BAC at pre-engineered chromosomal sites. This technique involved retroviral transduction of acceptor loci, containing an HSV thymidine kinase marker, and subsequent integration of BAC constructs into the acceptor sites, utilizing the loxP and lox511 sites present in the vector backbones. The BAC reporter, with a Renilla luciferase cassette inserted downstream of the hTERT promoter, was retrofitted with a puromycin marker. Through puromycin selection and ganciclovir counter-selection, a targeting efficiency of over 50% was achieved. We demonstrated that the activity and chromatin structures of the hTERT promoter in chromosomally integrated BAC reporter recapitulated its endogenous counterpart of the host cells. Therefore, we have established a genetically amendable platform to study chromatin and epigenetic regulation of the hTERT gene. The highly efficient and versatile RMBT technique has general applicability for studying largely unexplored chromatin-dependent mechanisms of promoter regulation of various genes.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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.