Strict control of telomerase activation using Cre-mediated inversion

BMC Biotechnology
Mark D Ungrin, Lea Harrington

Abstract

Human cells appear exquisitely sensitive to the levels of hTERT expression, the telomerase reverse transcriptase. In primary cells that do not express hTERT, telomeres erode with each successive cell division, leading to the eventual loss of telomere DNA, an induction of a telomere DNA damage response, and the onset of cellular senescence or crisis. In some instances, an average of less than one appropriately spliced hTERT transcript per cell appears sufficient to restore telomerase activity and telomere maintenance, and overcome finite replicative capacity. To underscore this sensitivity, we showed that a widely used system of transcriptional induction involving ecdysone (muristerone) led to sufficient expression of hTERT to immortalize human fibroblasts, even in the absence of induction. To permit tightly regulated expression of hTERT, or any other gene of interest, we developed a method of transcriptional control using an invertible expression cassette flanked by antiparallel loxP recombination sites. When introduced into human fibroblasts with the hTERT cDNA positioned in the opposite orientation relative to a constitutively active promoter, no telomerase activity was detected, and the cell population retained a mortal phen...Continue Reading

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Citations

Apr 7, 2007·Transgenic Research·Frank Schnütgen, Norbert B Ghyselinck
Sep 10, 2008·Proceedings of the National Academy of Sciences of the United States of America·Patrick SternRichard O Hynes
Apr 26, 2007·Academic Medicine : Journal of the Association of American Medical Colleges·Grace HuangChris Candler
Sep 28, 2013·Academic Medicine : Journal of the Association of American Medical Colleges·Valerie J LangDario Torre
Jul 4, 2006·Experimental Gerontology·Helen B Fleisig, Judy M Y Wong

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Datasets Mentioned

BETA
AF015950

Methods Mentioned

BETA
fluorescence activated cell sorting
flow cytometry
PCR
transfection
electrophoresis

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