Consecució i validació del silenciament de ST3GAL4 en línies cel·lulars de càncer de pàncrees: optimització de la metodologia

Abstract

Cancer is caused by a disproportionate division and growth of cells that end up forming a tumour (due to changes in the expression of oncogenes and tumour suppressor genes). It goes through different stages, the last of which is metastasis. Metastasis occurs when some tumour cells are able to travel through lymphatic or blood vessels and end up spreading in another part of the body. One of the cancers with the highest mortality is pancreatic ductal adenocarcinoma (PDAC). It has been shown that the glucidic antigen sialyl Lewis x (sLex) plays an important role in the adhesion of tumour cells to the endothelium. Usually, it is overexpressed in tumour cells because of the overexpression of the sialyltransferase ST3GAL IV (which participates in the antigen biosynthesis). The silencing of the ST3GAL4 gene could decrease the metastatic capacity of PDAC cells. The CRISPR-Cas9 methodology allows gene edition of specific genome sites using the endonuclease Cas9 and an sgRNA complementary to the target sequence where the edition is to be performed. Specific gene editing using this method can be used more routinely than other existing systems such as ZFNs or TALENs. In order to carry out this work, I have collaborated in the project of achieving and validating the silencing of ST3GAL4 using the CRISPR-Cas9 methodology, optimizing and preparing some processes and steps within the overall project. Very diverse methods have been used, such as primer design, plasmid extraction, survival assays to blasticidin or gene editing detection systems based on the formation of heteroduplexes. Finally it has been possible to accomplish all the proposed objectives: the presence of the sgRNAs targets in the products amplified by the designed primers have been verified, lentiviral plasmids have been validated and extracted in order to perform gene silencing, the adequate concentration of blasticidin has been found in order to select the cells that have integrated Cas9 in its genome with this antibiotic, and it has been described that the electrophoretic mobility assay of heteroduplexes is better than the T7EI digestion in terms of the speed of the method