Determination of the complex cause and effect signalling pathways that lead from pluripotent precursor cells to differentiated tissue is a critical topic that impacts on the development of effective stem cell therapies. Notch signalling is important for many cell fate decisions, however for myogenic progenitors clear genetic proof is still required.
Because Notch mutants are embryonic lethal, we aim to develop a conditional inactivation system utilising Cre/lox murine transgenesis. We will introduce Cre recombinase expression under control of native Pax7 control elements as this gene defines the embryonic myogenic lineage and adult muscle satellite cells.
We will also use the Tet-On system to allow temporal control of Cre-mediated recombination. By crossing these Cre-expressing lines with mice carrying floxed alleles of Notch1 and its downstream effector RBPJk, we will be able to inactivate these genes in embryonic myogenic precursors and adult muscle satellite cells.
Whilst this transgenic approach is underway we will also conduct a collaborative in vitro side project whereby protein extract from cultured in vitro myoblasts are affinity captured to identify interactors for a previously identified critical E-box transcriptional binding site located in the Myf5 promoter.
Overall, our transgenic approach will provide the means to determine the role of Notch signalling in myogenic precursor cell development in the embryo and adult.
The Pax7 specific Cre expressing transgenics will also provide a tool that we will be able to apply to other gene targets in myogenesis, which will help resolve the pathways essential to myogenic cell fate determination. Additionally, we will be investigating potential new regulators of Myf5 in vitro.
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