Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

• Published in: Journal of visualized experiments no. 129
• Main Authors: Kubala, Marta H., DeClerck, Yves A.
• Format: Journal Article
• Language: English
• Published: United States MyJove Corporation 23.11.2017
• Subjects: Animals; Breast Neoplasms - genetics; Breast Neoplasms - pathology; Cancer Research; Cell Line, Tumor; Cell Movement - physiology; Colonic Neoplasms - genetics; Colonic Neoplasms - pathology; cytochrome P-450; experimental design; Female; gene expression regulation; Gene Knockdown Techniques - methods; gene overexpression; genes; HCT116 Cells; human cell lines; Humans; macrophages; Macrophages - pathology; monocytes; Monocytes - pathology; neoplasm cells; neoplasms; protein synthesis; RNA, Small Interfering - genetics; small interfering RNA; tetracycline; Tumor Microenvironment
• ISSN: 1940-087X; 1940-087X
• 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.