通过条件性敲低癌细胞系中的基因表达来研究单核细胞/巨噬细胞向肿瘤微环境的募集。
Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment.
作者信息
Kubala Marta H, DeClerck Yves A
机构信息
Division of Hematology, Oncology and Blood and Marrow Transplantation, Department of Pediatrics, University of Southern California; The Saban Research Institute of Children's Hospital Los Angeles;
Division of Hematology, Oncology and Blood and Marrow Transplantation, Department of Pediatrics, University of Southern California; The Saban Research Institute of Children's Hospital Los Angeles; Department of Biochemistry and Molecular Medicine, University of Southern California.
出版信息
J Vis Exp. 2017 Nov 23(129):56333. doi: 10.3791/56333.
siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system, and the tetracycline regulated gene expression systems. The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.
小分子干扰RNA（siRNA）和短发夹RNA（shRNA）介导的基因表达敲低（KD）方法是理解基因和蛋白质功能的宝贵工具。然而，如果目标蛋白质的敲低对细胞具有致死作用，或者敲低的预期效果具有时间依赖性，那么无条件的敲低方法就不合适。在这些情况下，条件性系统更为适用，并且一直备受关注。这些系统包括蜕皮激素诱导过表达系统、细胞色素P-450诱导系统以及四环素调控基因表达系统。四环素调控基因表达系统能够通过在四环素存在的情况下诱导shRNA表达，实现对蛋白质表达的可逆控制。在本方案中，我们展示了一种在人类癌细胞系中使用功能性Tet-ON系统进行基因表达条件性调控的实验设计。然后我们证明了该系统在肿瘤细胞与单核细胞相互作用研究中的应用。
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