Over several years, Dr. Merlie has successfully collaborated with Dr. Sanes, applying molecular biology techniques to study development of the neuromuscular junction. In the recent past, common efforts of the two laboratories resulted in the generation of homologous recombination mouse mutants lacking s-laminin or 43k/rapsyn. Knock-out constructs for agrin and the AChR e gene have been introduced into ES cells. In this application, Dr. Merlie proposes to use cutting edge molecular technology to study the role of specific genes in neuromuscular junction formation. He lists the following Specific Aims: 1) To generate a skeletal muscle specific knock-out of the s-laminin gene using the Cre/lox P strategy. 2) To study the role of laminin B1 to s-laminin isoform transition in synapse formation by: (1) Rescue of the s-laminin knock out with transgenic constructs encoding s/B1 chimeras; and, (2) Ectopic expression of s-laminin. 3) To generate skeletal muscle and motoneuron specific knock-outs of agrin. 4) To study the role of AChR isoforms in synapse formation by: (1) Converting the g-subunit to an e-subunit and vice versa; and, (2) Rescue of e-gene knock-out with transgenic g/e chimeras to test the roles of extracellular, channel and intracellular domains.
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