p300-mediated gene expression during muscle cell survival

Type of experiment: The primary focus of these studies is to define the transcriptional network regulated by ectopic expression of the transcriptional co-activator protein, p300, in order to define its mechanism(s) of action in promoting muscle cell survival. Experimental factors: Our laboratory has generated a murine C2-derived myoblast cell line stably expressing an IGF-II cDNA in antisense orientation (C2AS12 cells). These cells proliferate normally in serum-rich growth medium but progressively die in low-serum differentiation medium. Ectopic expression of p300 (a transcriptional co-coactivator with acetyltransferase activity) prevents the progressive cell death induced by serum withdrawal, however, the mechanism of this action is not understood. Further, over-expression of a mutated form of p300, lacking at protein interaction domain (deltaTAZ2), failed to maintain cell viability although it retains catalytic activity. Wild-type and mutant p300 are delivered using recombinant adenoviruses (Ad-p300 and Ad-p300deltaTAZ2) and their expression is regulated by a second recombinant adenovirus encoding the tetracycline transactivator protein (Ad-tTA), affording regulated expression of p300 forms (Tet-off system) and providing a control for viral load. Using these reagents the overall experiment was as follows: Three parallel series of C2AS12 cells were infected with (1) wt p300 + tTA, (2) p300deltaTAZ2 +tTA, (3) wt p300 +tTA +doxycycline. Infected cells were grown to confluence followed by transfer to low-serum differentiation medium (T0). Cell were isolated at this point and following 24 (T24) hours incubation for RNA isolation. Companion dishes of cells were included for analysis by immunocytochemistry to ensure regulated transgene expression and cell viabilities. Keywords: time course 6 biological replicates, representing 3 treatment groups, two timepoints and 2 biological outcomes (cell survival with p300wt and apoptotic cell death with p300wt+Dox and p300mut)

实验类型：本系列研究的核心目标为解析转录共激活蛋白p300（p300）异位表达所调控的转录网络，以阐明其促进肌细胞存活的作用机制。
实验要素：本实验室构建了一株稳定表达反义方向胰岛素样生长因子II cDNA（IGF-II cDNA）的小鼠C2来源成肌细胞系（C2AS12细胞）。该细胞在血清丰富的生长培养基中可正常增殖，但在低血清分化培养基中会逐渐发生死亡。异位表达p300（一种具备乙酰转移酶活性的转录共激活因子）可阻断血清剥夺诱导的进行性细胞死亡，但其具体作用机制尚未明确。此外，过表达缺失蛋白相互作用结构域（ΔTAZ2）的p300突变体，尽管该突变体仍保留催化活性，却无法维持细胞活力。
基因递送方式：野生型与突变型p300均通过重组腺病毒（recombinant adenovirus）进行递送，分别为Ad-p300与Ad-p300ΔTAZ2；其表达由另一株编码四环素反式激活蛋白（tetracycline transactivator protein）的重组腺病毒Ad-tTA调控，以此实现p300变体的可诱导表达（Tet-off系统），同时可作为病毒载量的对照。
整体实验方案：基于上述试剂，本实验设计如下：将三平行组的C2AS12细胞分别感染（1）野生型p300 + tTA、（2）p300ΔTAZ2 + tTA、（3）野生型p300 + tTA + 多西环素（doxycycline）。感染后的细胞培养至汇合状态，随后转移至低血清分化培养基中（记为T0时刻）。分别在该时刻及培养24小时（T24）后收集细胞，用于RNA提取。同时设置配套细胞培养板，通过免疫细胞化学（immunocytochemistry）分析验证转基因的调控表达情况与细胞活力。
关键词：时间进程；6个生物学重复，涵盖3个处理组、2个时间点与2类生物学结局：p300野生型组对应细胞存活，p300野生型+多西环素组与p300突变体组对应细胞凋亡。