The mouse provides an invaluable mammalian model for skeletal muscle research, partly due to continuous development of sophisticated genetic tools. The tamoxifen (tmx) inducible forms of Cre DNA recombinase (Cre) - estrogen receptor (ER) fusion protein is a powerful tool for inducible gene manipulation. A complete set of myogenic Cre-ERT2 (CE) alleles should facilitate the progress of the field. We have initiated making a series of myogenic CE KI alleles at the Pax3, Myf5, MyoD, Mrf4, Myogenin (Mgn), and Myosin light chain 1f (Mlc1f) loci (Aim 1). Together with Pax7, their expression represents a sequence of events, from muscle progenitor to terminally differentiated state, during development and regeneration. These new alleles will not only be useful for our research, but also beneficial to researchers in the field at large. There have been data implicating cell sources that do not express Pax7 (Pax7- cells) but act as muscle stem cells (e.g. CD45+Sca1+ cells, PICs, or pericytes). We hypothesize that these proclaimed Pax7- muscle stem cells are in fact myogenic cells that loses Pax7 expression in transit to differentiation but can regain Pax7 expression and return to the muscle stem cell state (Aim 2).
Aim 1 : Generating and characterizing a myogenic series of CE alleles. This series is under various stages of development. They will be used to perform tmx-inducible cell marking. Short-term cell marking will be performed to characterize the specificity of cell marking and the cell potential to contribute to muscles and other cell types.
Aim 2 : Testing the possibility that differentiating myogenic cells can revert to the muscle stem cell state. Two scenarios will be tested: 1) developmental progression and 2) regeneration. We will determine whether certain CE lines that do not label Pax7+ cells in short-term labeling, but give rise to Pax7+ cells after long term tracing, in either experimental paradigm. While the """"""""reverted"""""""" stem cell population hypothesized may only represent a minor fraction, they likely have the potential to replenish the Pax7+ cells over time in chronic muscle wasting diseases and during the aging process.
To facilitate research in the skeletal muscle field, we propose to generate six new Cre-ERT2 mouse strains for inducible gene manipulation in the animal model. This genetic tool set should greatly enhance our ability to study muscle stem cell behavior as well as test gene function in skeletal muscles during normal development and injury induced regeneration. In principle, they can be used to study similar issues in chronic muscle wasting diseases and muscle aging.
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