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 μg 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.