"Reversibility of Differentiating Myogenic Cells to Muscle Stem Cells"

描述(由申请人提供)：小鼠为骨骼肌研究提供了一个无价的哺乳动物模型，部分原因是复杂的遗传工具的不断发展。三苯氧胺(TMX)诱导形式的Cre DNA重组酶(Cre)-雌激素受体(ER)融合蛋白是进行诱导基因操作的有力工具。一套完整的肌源性Cre-ERT2(CE)等位基因应该会促进这一领域的进展。我们已经开始在Pax3、Myf5、MyoD、MRf4、Mygenin(MGN)和肌球蛋白轻链1f(Mlc1f)基因座上制造一系列肌源性CE Ki等位基因(目标1)。与Pax7一起，它们的表达代表了一系列事件，从肌肉前体到终末分化状态，在发育和再生过程中。这些新的等位基因不仅对我们的研究有用，而且对该领域的研究人员也是有益的。已经有数据表明，细胞来源不表达Pax7(Pax7细胞)，但充当肌肉干细胞(例如CD45Sca1细胞、PIC或周细胞)。我们假设这些已宣布的Pax7-肌肉干细胞实际上是肌源性细胞，在向分化的过程中失去了Pax7的表达，但可以重新获得Pax7的表达，并返回到肌肉干细胞状态(目标2)。目的1：产生和表征一系列肌源性CE等位基因。本系列正处于不同的开发阶段。它们将被用于进行TMX诱导的细胞标记。将进行短期细胞标记，以表征细胞标记的特异性和对肌肉和其他细胞类型做出贡献的细胞潜力。目的2：检测分化中的肌源性细胞能否恢复到肌肉干细胞状态。将测试两种情景：1)发育过程和2)再生。我们将确定某些CE系在短期标记中不标记Pax7细胞，但在长期追踪后是否在任一实验范式中产生Pax7细胞。虽然假设的“逆转”干细胞群体可能只是一小部分，但随着时间的推移，它们很可能在慢性肌肉衰减性疾病和衰老过程中补充Pax7细胞。

DESCRIPTION (provided by applicant): 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.