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The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals.

Abstract

BACKGROUND

The reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity.

RESULTS

Three constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells.

CONCLUSION

The 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues.

Authors+Show Affiliations

Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Madrid 28040, Spain. pcamacho@inia.esNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16457732

Citation

Pericuesta, Eva, et al. "The Proximal Promoter Region of mTert Is Sufficient to Regulate Telomerase Activity in ES Cells and Transgenic Animals." Reproductive Biology and Endocrinology : RB&E, vol. 4, 2006, p. 5.
Pericuesta E, Ramírez MA, Villa-Diaz A, et al. The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals. Reprod Biol Endocrinol. 2006;4:5.
Pericuesta, E., Ramírez, M. A., Villa-Diaz, A., Relaño-Gines, A., Torres, J. M., Nieto, M., ... Gutiérrez-Adán, A. (2006). The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals. Reproductive Biology and Endocrinology : RB&E, 4, p. 5.
Pericuesta E, et al. The Proximal Promoter Region of mTert Is Sufficient to Regulate Telomerase Activity in ES Cells and Transgenic Animals. Reprod Biol Endocrinol. 2006 Feb 3;4:5. PubMed PMID: 16457732.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals. AU - Pericuesta,Eva, AU - Ramírez,Miguel Angel, AU - Villa-Diaz,Ana, AU - Relaño-Gines,Aroa, AU - Torres,Juan Maria, AU - Nieto,Marta, AU - Pintado,Belen, AU - Gutiérrez-Adán,Alfonso, Y1 - 2006/02/03/ PY - 2005/11/18/received PY - 2006/02/03/accepted PY - 2006/2/7/pubmed PY - 2006/4/1/medline PY - 2006/2/7/entrez SP - 5 EP - 5 JF - Reproductive biology and endocrinology : RB&E JO - Reprod. Biol. Endocrinol. VL - 4 N2 - BACKGROUND: The reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity. RESULTS: Three constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells. CONCLUSION: The 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues. SN - 1477-7827 UR - https://www.unboundmedicine.com/medline/citation/16457732/The_proximal_promoter_region_of_mTert_is_sufficient_to_regulate_telomerase_activity_in_ES_cells_and_transgenic_animals_ L2 - https://rbej.biomedcentral.com/articles/10.1186/1477-7827-4-5 DB - PRIME DP - Unbound Medicine ER -