Studying human telomerase gene transcription by a chromatinized reporter generated by recombinase-mediated targeting of a bacterial artificial chromosome
Open Access
- 15 June 2009
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 37 (17), e111
- https://doi.org/10.1093/nar/gkp511
Abstract
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.Keywords
This publication has 44 references indexed in Scilit:
- Differential repression of human and mouse TERT genes during cell differentiationNucleic Acids Research, 2009
- A New Positive/Negative Selection Scheme for Precise BAC RecombineeringMolecular Biotechnology, 2009
- Mosaic Complementation Demonstrates a Regulatory Role for Myosin VIIa in Actin Dynamics of StereociliaMolecular and Cellular Biology, 2008
- Altered Histone H1 Stoichiometry and an Absence of Nucleosome Positioning on Transfected DNAPublished by Elsevier BV ,2008
- Transcriptional Silencing of a Novel hTERT Reporter Locus during In Vitro Differentiation of Mouse Embryonic Stem CellsMolecular Biology of the Cell, 2007
- Transcription regulation and animal diversityNature, 2003
- Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activityInternational Journal of Cancer, 2002
- Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem CellsMolecular and Cellular Biology, 2001
- Segmental genomic replacement in embryonic stem cells by double lox targetingNucleic Acids Research, 1999
- A 5′ element of the chicken β-globin domain serves as an insulator in human erythroid cells and protects against position effect in DrosophilaCell, 1993