760. Development of an HSV-Based Model System to Identify Factors Acting in Embryonic Myogenesis

Abstract

Top of pageAbstract Myogenesis is a complex process involving several distinct pathways in the ordered proliferation, migration, and differentiation of stem cells in the developing embryo. The cascade of gene regulation governing embryonic myogenesis has significant gaps of information which has prompted the development of a model system to fill these gaps using herpes simplex virus vectors. We use murine embryonic stem cells (mES) because their pluripotent characteristics provide an ideal model for differentiation along myogenic lineages. We are particularly interested in the identification of genes active during early stages of myogenesis, prior to the activation of basic helix-loop-helix (bHLH) muscle transcription factors MyoD, Myf5, myogenin, and MRF4. Pax3 is our initial target as it has been shown to activate gene expression of the bHLH muscle markers and induce myogenesis in certain pluripotent stem cells and embryonic tissues. Since activators of Pax3 have not been identified, we have begun exploring the possibility of using a mutant HSV-1 viral vector, JD|[beta]||[beta]|, to study early promoter-activating events of myogenesis. JD|[beta]||[beta]| is deleted for ICP4 and ICP22, ICP0 and ICP27 have been converted into |[beta]| (early) genes, and one full genomic repeat element has been eliminated. Preliminary flow cytometry data show that JD|[beta]||[beta]| drives robust reporter transgene expression in embryonic stem cells and is non-toxic. Characterization of this virus, including toxicity studies, transgene expression levels, and IE gene kinetics, will be reported. Several mES cell lines containing myogenic marker genes have been developed by others and characterized, including a Pax3-lacZ mES cell line. mES cells, differentiated into muscle using ectopic differentiating methods, serve as a control. JD|[beta]||[beta]| will be modified to carry genes potentially involved during early myogenesis including |[beta]|-catenin and Wnt3a. In order to ascertain whether these or other transgenes can functionally activate the Pax3 promoter, we will deliver them to Pax3-lacZ mES cells and assay for reporter gene expression as well as activation of downstream muscle-specific marker genes. This will allow us to validate the use of HSV as a means to introduce transgenes in mES cells and assay for muscle differentiation as a result of transgene expression. Upstream factors with consistent, ordered activity as determined by the proposed model system are of great interest for deciphering stages of myogenesis, and will be potential candidates for committing early progenitor cells into the muscle phenotype for therapeutic purposes.