Application of the CRISPR Cas-9 technique to obtain a hepatic bovine in vitro model (BFH12) deleted of the CYP1A1 and CYP3A28 genes

Bovine is one of the most relevant veterinary species and one of the most essential sources of food in the industrialized world. Being cattle one of the most prevalent species in the food sector -used for meat products, milk and cheese- interest is beginning to arise as to how these animals can metabolize xenobiotics (i.e., drugs, natural toxins and environmental contaminants) to which they are exposed and the possible presence of residues in foodstuff. Cytochrome P450 super family is responsible for most of the phase I reactions of endogenous and exogenous compounds, that mainly take place in the liver. These reactions could result in the biotransformation or in the bioactivation of xenobiotics. In the first case, the metabolites resulting from the CYPs mediated biotransformation are less toxic compared to the parental compound, whereas in case of bioactivation the products are more toxic than the original compound. The aim of this work was to create an in vitro model derived from the BFH12 cell line, a bovine fetal hepatocytes cell line, deleted of the CYP1A1 and CYP3A28 genes through the application of the CRISPR Cas-9 technique. These two genes were chosen due to their importance in the metabolism of xenobiotics, as mentioned before. The end goal was to create two knockout cell lines, one deleted of CYP1A1 and one deleted of CYP3A28, and one showing the double knockout, to carry out an in-depth investigation of the involvement of each isoform in bovine hepatic metabolism and the biological consequences. Once the knockout cell lines were obtained, some confirmatory studies were performed to characterize CYP1A1 and CYP3A28 expression in the clones by means of immunoblotting and/or catalytic activity.