Experimental Strategies to Manipulate the Cellular Levels of the Multifunctional Factor CTCF
Cellular homeostasis is the result of an intricate and coordinated combinatorial of biochemical and molecular processes. Among them is the control of gene expression in the context of the chromatin structure which is central for cell survival. Interdependent action of transcription factors, cofactors, chromatin remodeling activities, and three-dimensional organization of the genome are responsible to reach exquisite levels of gene expression. Among such transcription factors there is a subset of highly specialized nuclear factors with features resembling master regulators with a large variety of functions. This is turning to be the case of the multifunctional nuclear factor CCCTC-binding protein (CTCF) which is involved in gene regulation, chromatin organization, and three-dimensional conformation of the genome inside the cell nucleus. Technically its study has turned to be challenging, in particular its posttranscriptional interference by small interference RNAs. Here we describe three main strategies to downregulate the overall abundance of CTCF in culture cell lines.
Key wordsCTCF Lentivirus Interference RNA Inducible vectors Lipofection Electroporation Fluorescent cytometry
We acknowledge Ricardo Saldaña-Meyer for comments and critical reading of the manuscript. This work was supported by the Dirección General de Asuntos del Personal Académico-Universidad Nacional Autónoma de México (IN209403 and IN203811), Consejo Nacional de Ciencia y Tecnología, México (CONACyT; 42653-Q and 128464), and a Ph.D. fellowship from CONACyT and Dirección General de estudios de Posgrado-Universidad Nacional Autónoma de México (DGEP) (EG-B: 207989, RP-M: 173754, and EA-O: 256339). Additional support was provided by the Ph.D. Graduate Program, “Doctorado en Ciencias Biomédicas and Ciencias Bioquímicas.” We thank Fernando Suaste-Olmos for technical assistance.
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