The overall goal of this project is to employ a novel functional genomics approach to identify geneswhose 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 ofthe immediate early (IE) genes and capable of replication in cells engineered to contain avector-inducible, trans-complementing ICP4 gene.
In Aim 2, a panel of mouse ES cell lines will be createdin which either the EGFP or ICP4 gene will be recombined into the cellular genome juxtaposed to thepromoter that drives expression of one of the muscle differentiation factors Pax7, myf5, or myoD. Usingestablished culture conditions, the EGFP cell lines will be differentiated towards muscle cells and theappearance 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 arecombination system, 'Gateway' (Invitrogen), which will then be used to introduce a mouse musclelprogenitor cell cDNA library into the vector. The vector library will be propagated in bacteria as singlelcopy plasmids, its complexity established by quantitative PCR for low and medium copy genes andltransfected into ICP4-complementing Vero cells for virus production.
In Aim 4, pooled library vectors willlbe propagated on complementing Vero cells in microtiter wells and replica plated onto the three myogenicpromoter-ICP4 ES cell lines. Progeny vector particles generated in cells expressing ICP4 by induction ofthe resident myogenic promoter will be isolated by limiting dilution on complementing Vero cells andrescreened on the panel of EGFP ES cell lines to determine whether the cellular factor is functional inactivating early and late stage myogenic factor promoters. The cDNA of activating vectors will besequenced, compared with known data bases for preliminary assessment of function and furthercharacterized 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 musclecell development.
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