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


CONCLUSIONS
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.
Pericuesta
Eva
E
Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Madrid 28040, Spain. pcamacho@inia.es
Ramírez
Miguel Angel
MA
Villa-Diaz
Ana
A
Relaño-Gines
Aroa
A
Torres
Juan Maria
JM
Nieto
Marta
M
Pintado
Belen
B
Gutiérrez-Adán
Alfonso
A
D000818:Animals; D001921:Brain / Q000196:embryology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013234:Stem Cells / Q000502:physiology*; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000502:physiology
D004268:DNA-Binding Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C509186:TERT protein, human; D019098:Telomerase; C509187:Tert protein, mouse