Development of transgenic knockdown methods in Ambystoma mexicanum to study satellite cell activation during limb regeneration

蝾螈附肢再生诱导成体组织产生具有胚胎特性的祖细胞，称为芽基。这可以通过成熟细胞类型的去分化或驻留干细胞的激活来发生。去分化与卫星干细胞活化对再生的相对重要性是完全未知的。在这里，我们将确定肌肉去分化与卫星细胞激活的作用，在附肢再生作为一个模型系统的蝾螈。我们将分别使用cre/loxP介导的重组转基因动物中的去分化肌肉和卫星细胞的命运。我们以前已经开发了转基因的蝾螈，并证明了cre/loxP系统的功能，有效地在成熟的组织。我们将通过抑制其发生来进一步测试去分化相对于干细胞的作用。为了有效地做到这一点，我们将首先开发位点特异性整合到蝾螈基因组中，这样我们就不必在进行实验之前将转基因通过种系。然后，我们将整合表达盒，永久表达肌肉分化因子，肌细胞生成素，和细胞周期抑制剂，p16 INK 4在肌肉或卫星细胞，以阻止生产的祖细胞从这些来源。最后，我们的目标是剖析控制过程的分子途径，如肌肉脱分化，参与早期芽基形成使用这些整合策略。我们的第一个目标是wnt通路。

Salamander appendage regeneration induces adult tissue to produce progenitor cells with embryonic properties called the blastema. This may occur by dedifferentiation of mature cell types or activation of resident stem cells. The relative importance of dedifferentiation versus satellite stem cell activation to regeneration is completely unknown. Here we will determine the role of muscle dedifferentiation versus satellite cell activation in appendage regeneration using the axolotl as a model system. We will separately track the fate of dedifferentiated muscle and satellite cells using cre/loxP mediated recombination in transgenic animals. We have previously developed transgenesis in the axolotl and demonstrated that the cre/loxP system functions efficiently in mature tissues. We will further test the role of dedifferentiation versus stem cells by inhibiting their occurrence. To do this efficiently we will first develop site-specific integration into the axolotl genome so that we do not have to pass trangenes through the germline before performing experiments. We will then integrate expression cassettes to permanently express the muscle differentiation factor, myogenin, and the cell cycle inhibitor, p16INK4 in muscle or satellite cells to block production of progenitors from these sources. Finally our goal is to dissect the molecular pathways controlling processes like muscle dedifferentiation that are involved in early blastema formation using these integration strategies. Our first target will be the wnt pathway.