177 GENERATION OF ES CELLS AND TRANSGENIC MICE EXPRESSING MTERT-GFP AS A MARKER OF PLURIPOTENTIAL CELLS

Abstract

There is not a simple system that allows us to identify stem cells in adult tissues. Cells of adult tissues arise from dividing progenitor cells, which themselves are derived from multipotential stem cells. Telomerase is the enzyme that maintains the ends of linear chromosomes in eukaryotic cells. Recently, a segment of the promoter sequence of the reverse transcriptase of murine telomerase (mTert) has been characterized. mTert is expressed with greatest abundance during embryogenesis and becomes widely expressed in adult tissues at low levels. This low expression level in adult tissues may be due to the presence of pluripotent stem cells present in those tissues. To examine the relationship between telomerase activity and multipotential of adult cells we have generated three constructs (1k-, 2k-, and 5k-mTert-GFP) comprising different segments of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (GFP). These constructs were electroporated into R1 and B6D2 (generated in our laboratory) ES cells and were used to produce transgenic mice. The generation and identification of transgenic mice (C57BL6 × CBA) has been previously described (Gutierrez-Adan and Pintado 2000 Trangenic Res. 9, 81–89). Transgenic founders were backcrossed to C57BL6 × CBA mice to obtain transgenic lines. The three constructs were able to mimic the mTert expression, which was coupled to green fluorescence. The mTert-GFP transfected ES cells were initially maintained in medium supplemented with LIF, which was subsequently removed to allow differentiation of embryoid bodies (EBs) and other cell types. GFP expression was higher during the first two days after LIF removal (period of enhanced cell proliferation), decreasing in the following days as a result of EB differentiation. Both ES cell lines showed reduced GFP expression upon differentiation, suggesting that mTert is the principal determinant of telomerase activity; moreover, different degrees of expression and down regulation were reported with the different constructs. Using these constructs we have also generated transgenic mice. Eight lines of transgenic mice carrying the 1kmTert-GFP transgene, four with the 2kmTert-GFP, and three with the 5kmTert-GFP, were obtained. There were no significant differences between the proportions of transgenic founder generates. The transgenic mice express and GFP during the fetal development, indicating their telomerase activity. We are now analyzing the expression of mTert-GFP in adults tissues. Our results suggest that telomerase-GFP transgenics are an important tool to assess the role of telomerase in adult multipotential cells as well as to select these pluripotent cells in adult tissue. It will be interesting to see if different levels of mTert-GFP expression are associated with different levels of pluripotency.