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DOI: 10.3791/58222-v
Anna M. Chiarella1, Tiffany A. Wang2, Kyle V. Butler3, Jian Jin3, Nathaniel A. Hathaway4
1Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology,University of North Carolina, 2College of Arts and Sciences, Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery,University of North Carolina, 3Chemical Biology and Drug Discovery, Pharmacological Sciences and Oncological Sciences,Icahn School of Medicine at Mount Sinai, 4Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center,University of North Carolina
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This article describes a method for controlling gene expression by recruiting chromatin-modifying machinery in a gene-specific and reversible manner. This technique addresses key questions in the epigenetics field, particularly regarding gene regulation mechanisms.
Reglamento de medio ambiente de la cromatina es un proceso esencial para expresión génica adecuada. Aquí, describimos un método para controlar la expresión génica a través de la contratación de modificadores de la cromatina maquinaria de manera gene-específica y reversible.
Este método puede ayudar a responder preguntas clave en el campo de la epigenética, como los mecanismos de regulación genética y las relaciones de causa y efecto entre los diferentes elementos reguladores de la cromatina. La principal ventaja de esta técnica es que por primera vez es posible módulor un loci de cromatina con enzimas endógenas fisiológicamente relevantes. Generalmente los individuos nuevos en este método lucharán porque la cultura de LA ESL del ratón es inherentemente difícil para aquellos que no han sido entrenados en la técnica.
La demostración adicional de este método es fundamental, ya que los pasos de infección viral celular son difíciles de aprender. El investigador debe cronolar dos líneas separadas de cultivo celular y es necesario vigilar vigilantemente la seguridad durante estos pasos. Para comenzar el protocolo, pasaje 18 millones de células HEK 293T por placa de 50 centímetros.
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