title: The use of real-time PCR with fluorogenic probes for the rapid selection of mutant neuroectodermal grafts.
abstract: Adenosine is an efficient inhibitor of neuronal activity with the ability to suppress seizure activity in various animal models of epilepsy. In the present study adenosine-releasing neuronal cells were generated as a potential source for therapeutically active grafts. Mice with a genetic disruption of the gene encoding adenosine kinase (Adk(-/-))-the major adenosine metabolizing enzyme-were used as a source for the derivation of adenosine releasing neuronal cells. Since homozygous Adk(-/-) mice constitute a lethal phenotype, embryonic neuroectoderm was derived from intercrosses of Adk(+/-)-mice. Therefore, a rapid genotyping procedure had to be developed using a fluorescent 5'-exonuclease (TaqMan) assay, which permitted the genotyping of embryonic cell material within 3 h. During this time period the cells to be grafted displayed an unaltered viability. Cultured neuroectodermal Adk(-/-) cells released up to 2 micro g adenosine per mg protein per hour. Adk(-/-) neuroectoderm grafted into the lateral brain ventricle of adult mice was found to survive for at least 6 weeks. The method described here suggests the feasibility to graft adenosine releasing neuroectodermal cells as a potential therapeutic approach for the treatment of pharmacoresistant epilepsy.
mesh_terms: D000248:Adenosine Kinase / Q000172:deficiency* / Q000235:genetics; D000818:Animals; D016380:Brain Tissue Transplantation / Q000379:methods*; D002478:Cells, Cultured; D004475:Ectoderm / Q000637:transplantation*; D004622:Embryo, Mammalian; D005456:Fluorescent Dyes; D005838:Genotype; D020547:Lateral Ventricles / Q000637:transplantation; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D009474:Neurons / Q000637:transplantation*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019737:Transplants
chemical_list: D005456:Fluorescent Dyes; D000248:Adenosine Kinase