Adult stem cells from multiple tissues express telomerase, which is down-regulated upon differentiation. Identifying these telomerase expressing cells may facilitate the isolation and characterization of adult stem cells from multiple tissues. Using a well characterized reporter gene construct, mTert:GFP (kindly provided by Dr. N. Hole, Durham University, UK) we generated transgenic ES cells and selected clones expressing GFP at high levels. To screen against genomic integration events resulting in constitutive GFP expression we next subjected each clone to embryoid body (EB) formation as a model of in vitro differentiation. Using ES cells in which Tert GFP was expressed in the ES cell but not embryoid bodies, three lines of transgenic mice were generated and bred to germ-line heterozygosity. These mice are currently being studied to validate whether GFP(+) cells, expressed in various stem cell compartments (bone marrow, testis, gut), are in fact stem cells.Analyses of sorted bone marrow cells from two transgenic mouse lines have demonstrated GFP expression within long-term hematopoeitic stem cells. These results have been further validated using serial transplantation of GFP(+) bone marrow cells. In addition, both lines express GFP within testicular germ cells and within small intestinal crypt cells. Once these initial validation studies are complete, the brain will be serially sectioned at various stages of development to identify GFP(+) cells. In addition, we have generated neurospheres from mTert:GFP embryonic brain regions and are currently characterizing their GFP expression profile. Future studies will be aimed at characterizing GFP expression profiles under various conditions of neuronal development in collaboration with DDRC investigators. Our studies of adrenal gland development have led us to disrupt the aldosterone synthase (AS) gene locus in mice. Breeding AS-Cre knock out mice to homozygosity results in Aldosterone Synthase knock-out mice (ASKO). Aldosterone deficiency results in a classical form of congenital salt-wasting adrenal hyperplasia and is associated with significant perinatal morbidity and mortality if left untreated. In addition, the syndrome of aldosterone deficiency gives rise to a growth restriction phenotype due to an unknown mechanism. Also, AS is expressed in the hippocampus, where it has an unknown function and may be involved in the physiologic stress responses and/or food/H20/salt seeking behavior(s). We will study the effects of AS deficiency on hippocampal development and on learning and memory using AS knock-outs.
