An investigation into whether telomerase can be used to identify stem cells in the mouse

Telomeres are G-rich strands of DNA that protect chromosome ends from recognition as a DNA break. Telomeric DNA addition is catalysed by the reverse transcriptase telomerase which consists of a catalytic component; Tert and an RNA component Terc. In vitro and in vivo studies demonstrated that the primary mode of regulation of telomerase is through transcriptional regulation of the Tert gene. The mTert promoter driving a fluorescent reporter can recapitulate endogenous mTert expression in ES cells in vitro. Telomerase activity is a characteristic of stem cells. However, upon differentiation of stem cells, telomerase activity is switched off; resulting in the progressive shortening of telomeres until they are critically short and cellular senescence is triggered. Given that telomerase activity is determined by Tert promoter activity, it is hypothesised here that the Tert promoter could be used to mark stem cells in the adult.;A vector was generated in which the mTert promoter drives the expression of CFP. In vitro analysis of Tert-CFP ES clones revealed that CFP was expressed in undifferentiated cells but was down-regulated as ES cells were differentiated. Teratomas were generated from ES cells expressing the transgenic vector and CFP expression was found to correlate with regions of undifferentiation and intense proliferation.;Direct DNA microinjection generated four transgenic mouse lines containing the transgene within the germline. RT-PCR for endogenous mTert and CFP showed that their expression varied similarly from two transgenic lines. Histological analysis of testis and skin has shown expression in spermatogonia, basal layer and bulge cells, thus indicating activity of the reporter in these stem cells. However, reporter expression is also evident in spermatocytes of the testis and spinous and granular layers of the skin. Thus, the reporter not only identifies highly proliferative cells, it also identifies cells with a lower proliferative potential.