The overall goal of this project is to employ a novel functional genomics approach to identify genes whose products induce the differentiation of mouse embryonic stem (ES) cells towards muscle cells.
In Aim 1, a highly defective, non-cytotoxic HSV mutant will be developed that is deficient for expression of the immediate early (IE) genes and capable of replication in cells engineered to contain a vector-inducible, trans-complementing ICP4 gene.
In Aim 2, a panel of mouse ES cell lines will be created in which either the EGFP or ICP4 gene will be recombined into the cellular genome juxtaposed to the promoter that drives expression of one of the muscle differentiation factors Pax7, myf5, or myoD. Using established culture conditions, the EGFP cell lines will be differentiated towards muscle cells and the appearance of fluorescing cells will be correlated with detection of stage specific myocytic markers.
In Aim 3, the mutant virus will be engineered to contain a bacterial artificial chromosome and a recombination system, """"""""Gateway"""""""" (Invitrogen), which will then be used to introduce a mouse muscle lprogenitor cell cDNA library into the vector. The vector library will be propagated in bacteria as single copy plasmids, its complexity established by quantitative PCR for low and medium copy genes and ltransfected into ICP4-complementing Vero cells for virus production.
In Aim 4, pooled library vectors will be propagated on complementing Vero cells in microtiter wells and replica plated onto the three myogenic promoter-ICP4 ES cell lines. Progeny vector particles generated in cells expressing ICP4 by induction of the resident myogenic promoter will be isolated by limiting dilution on complementing Vero cells and rescreened on the panel of EGFP ES cell lines to determine whether the cellular factor is functional in activating early and late stage myogenic factor promoters. The cDNA of activating vectors will be sequenced, compared with known data bases for preliminary assessment of function and further characterized for their ability to induce muscle cell markers using immunological and biochemical assays. Ultimately, we hope to identify novel factors or signaling mechanisms that interact to control early muscle cell development.
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