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; Jiyue Zhu

doi:10.1093/nar/gkp511

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

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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

Nucleic acids research, Vol.37(17), pp.e111-e111

09/2009

DOI: https://doi.org/10.1093/nar/gkp511

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https://hdl.handle.net/2376/114220

PMCID: PMC2761251

PMID: 19528078

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Abstract

Cell Line

Gene Targeting - methods

Promoter Regions, Genetic

Humans

DNA (Cytosine-5-)-Methyltransferases - antagonists & inhibitors

Gene Expression Regulation, Enzymologic

Telomerase - genetics

Retroviridae - genetics

Chromosomes, Artificial, Bacterial

Integrases - metabolism

Transcription, Genetic

Genes, Reporter

Chromatin - chemistry

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.

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Title: Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome
Creators: Shuwen Wang - Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
Yuanjun Zhao
Melanie A Leiby
Jiyue Zhu
Publication Details: Nucleic acids research, Vol.37(17), pp.e111-e111
Academic Unit: Pharmaceutical Sciences, Department of
Publisher: England
Grant note: R01 GM071725 / NIGMS NIH HHS GM071725 / NIGMS NIH HHS
Identifiers: 99900547689401842
Language: English
Resource Type: Journal article