E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备
Summary
ACF染色质重塑因子E4orf4诱导的细胞死亡的贡献进行了测量。该协议包括在多西环素治疗诱导有条件击倒的ACF的亚基ACF1和SNF2h的细胞克隆的选择，使用的DAPI检测测量E4orf4-诱导的细胞死亡的诱导的细胞系。
Abstract
的研究是非常重要的贡献感兴趣的各种途径的1,2的蛋白质在哺乳动物细胞中的基因表达的功能失活。然而，有条件击倒的基因的表达是必需的情况下，当没有被细胞的耐受性，构击倒了很长一段的时间3-5。在这里，我们描述了一个协议，准备允许有条件的ACF染色质重塑因子亚基敲除的细胞株。这些细胞株诱导的细胞死亡的ACF的贡献便于确定由腺病毒E4orf4蛋白6。编码为ACF1和SNF2h亚基的ACF染色质重塑因子的短发夹RNA的序列进行克隆到一个多西环素诱导型启动子中还含有新霉素抗性基因的基因的质粒的旁边。选择的新霉素抗性细胞克隆在G418的存在下，和隔离。将所得的细胞系诱导的强力霉素治疗，而一旦ACF1或SNF2h表达水平降低，细胞转染的质粒编码E4orf4或空载体。确认具体的shRNA结构的影响，ACF1或SNF2h蛋白水平恢复到WT水平的转染与表达质粒ACF1或SNF2h呈现抵抗的shRNA引入沉默突变。通过DAPI染色的测定，其中的频率测定细胞核与细胞凋亡在转染的细胞群体的形貌的外观7-9 E4orf4的能力，诱导细胞死亡的各种样品中测定。
这里所描述的协议，可用于测定各种蛋白质的功能贡献的情况下诱导细胞死亡的蛋白质的合作伙伴，构击倒时，可能是电池致命的。
Protocol
Discussion
敲除特定基因的表达，是一个重要的贡献的一种蛋白质调控途径的调查方法。由于构击倒必需基因的表达产生负面影响细胞的增殖，他们的研究可能需要稳定的条件击倒系统的siRNA或应用程序无论是短暂的介绍。生成的稳定的细胞系的能力对于药物引起的这里描述的shRNA表达，提供了一个优势，在许多细胞系中，这可能不是高效的瞬时转染，并在使用的病毒，需要重复制备，并有时一个额外的短长…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是支持（部分）以色列科学基金会（批准11分之399），德意志研究联合会（DFG）的框架内，德国和以色列的项目合作（DIP）和拉巴波特家庭研究所的研究工作的医学科学。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| High glucose DMEM | Gibco | 41965 | |
| Tet system approved FBS | Clontech | 631106 | |
| L-glutamine | Gibco | 25030 | |
| Pen Strep | Gibco | 15140 | |
| 0.25% Trypsin-EDTA | Gibco | 25200 | |
| T-REx-293 cells | Invitrogen | R710-07 | |
| G418 | Sigma | A1720 | |
| blasticidin | Invitrogen | R210-01 | |
| pSuperior.neo+GFP plasmid | OligoEngine | VEC-PBS-0007/0008 | |
| jetPIE | Polyplus transfection | 101-40 | |
| doxycycline | Sigma | D9891 | |
| paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| 4′,6-diamidino-2-phenylindole (DAPI) | Sigma | D9542 | |
| Fluoromount-G | SouthernBiotech | 0100-01 |
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