Supresión del canal dependiente de volumen (VRAC) mediante la técnica shRNA en células C2C12 de Mus musculus
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Date
2021
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Universidad Nacional de Trujillo
Abstract
El presente trabajo de investigación tiene como objetivo generar vectores plasmídicos para la supresión de la expresión de los genes Lrrc8c y Lrrc8d que codifican para VRAC en la línea celular C2C12 de Mus musculus. El silenciamiento genético in vitro mediante shRNA (small hairpin RNA) ligado a un plásmido, es una técnica que suprime o modula la traducción de proteínas mediante la inhibición de un RNA mensajero (mRNA) diana. Los mRNA de los genes mencionados codifican para dos proteínas que forman parte de un canal de aniones regulado por volumen, que se expresa de forma constitutiva en diversas células de vertebrados jugando un papel clave en la regulación del volumen celular, así como en la secreción de fluidos, permeabilidad iónica y captación de determinados fármacos. Se emplearon diversas herramientas informáticas, las cuales permitieron construir una serie de shRNA, tres por cada gen, que poseen una cadena complementaria a secuencias específicas dentro de exones del mRNA de los genes Lrrc8c y Lrrc8d de Mus musculus. Los shRNA generados fueron ligados correctamente al plásmido PMKO.1 GFP y posteriormente transfectados en la línea celular muscular C2C12 de Mus musculus. Se concluye que se logró la transfección de dos plásmidos ligados a los shRNA, denominados: shRNA Lrrc8c FR1 GFP1 y shRNA Lrrc8d FR2 GFP2, cuya eficiencia para suprimir la expresión de los genes Lrrc8c y Lrrc8d fue de 32% y 17% respectivamente.
ABSTRACT The present research work aims to generate plasmid vectors for the suppression of the expression of the Lrrc8c and Lrrc8d genes that code for VRAC in the Mus musculus cell line C2C12. In vitro gene silencing using shRNA (small hairpin RNA) linked to a plasmid is a technique that suppresses or modulates protein translation by inhibiting a target messenger RNA (mRNA). The mRNAs of the aforementioned genes encode for two proteins that are part of a volume-regulated anion channel, which is expressed constitutively in various vertebrate cells, playing a key role in the regulation of cell volume, as well as in the secretion of fluids, ionic permeability and uptake of certain drugs. Various computer tools were used, which allowed the construction of a series of shRNAs, three for each gene, which have a complementary chain to specific sequences within exons of the mRNA of the Mus musculus genes Lrrc8c and Lrrc8d. The shRNAs generated were correctly ligated to the PMKO.1 GFP plasmid and subsequently transfected into the Mus musculus muscle cell line C2C12. It is concluded that the transfection of two plasmids linked to the shRNAs was achieved, named: shRNA Lrrc8c FR1 GFP1 and shRNA Lrrc8d FR2 GFP2, whose efficiency to suppress the expression of the Lrrc8c and Lrrc8d genes was 32% and 17% respectively.
ABSTRACT The present research work aims to generate plasmid vectors for the suppression of the expression of the Lrrc8c and Lrrc8d genes that code for VRAC in the Mus musculus cell line C2C12. In vitro gene silencing using shRNA (small hairpin RNA) linked to a plasmid is a technique that suppresses or modulates protein translation by inhibiting a target messenger RNA (mRNA). The mRNAs of the aforementioned genes encode for two proteins that are part of a volume-regulated anion channel, which is expressed constitutively in various vertebrate cells, playing a key role in the regulation of cell volume, as well as in the secretion of fluids, ionic permeability and uptake of certain drugs. Various computer tools were used, which allowed the construction of a series of shRNAs, three for each gene, which have a complementary chain to specific sequences within exons of the mRNA of the Mus musculus genes Lrrc8c and Lrrc8d. The shRNAs generated were correctly ligated to the PMKO.1 GFP plasmid and subsequently transfected into the Mus musculus muscle cell line C2C12. It is concluded that the transfection of two plasmids linked to the shRNAs was achieved, named: shRNA Lrrc8c FR1 GFP1 and shRNA Lrrc8d FR2 GFP2, whose efficiency to suppress the expression of the Lrrc8c and Lrrc8d genes was 32% and 17% respectively.
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Silenciamiento genético. Transfección. Transformación bacteriana. Reacción en cadena de la polimerasa