Produção de modelo murino para a doença de Alzheimer usando CRISPR/Cas9: deleção do enhancer BCR e construção de vetor para gene-targeting do alelo APOE4 humano sob controle do promotor hSYN no locus ROSA26 murino

Abstract

Alzheimer's Disease (AD) is a neurodegenerative disorder that affects millions of people worldwide. This is a still uncured and undiagnosed disorder and many aspects of its pathophysiology are unknown. For this reason, there is a great demand for an animal model that reproduces all the histopathological and behavioral findings of patients with AD. Many models have already been produced, but none of them recapitulate all aspects of this pathology. The main genetic risk factor known for AD is the presence of the APOE4 allele of the APOE gene in the genome of the individuals. This gene is regulated by a sequence called BCR, a neuro-specific enhancer, conserved in the human and murine genome. This work is part of a project that aims to produce a new murine model for AD. The final model will have, in its genome, the deletion of the BCR sequence and the insertion of the human APOE4 allele under the control of hSYN, a specific promoter for neurons present in the human genome. The objectives of this work were promote the deletion of BCR from mouse hepatocytes in vitro and produce a plasmid vector that enables the gene-targeting of hSYN and APOE4 in the murine genome at a locus that allows the stable expression of transgenes called ROSA26. The BCR deletion was performed with the support of gene editing system CRISPR/Cas9. Murine hepatocytes were transfected with a CRISPR/Cas9 system developed to promote BCR deletion and subsequently isolated and genotyped by PCR. For the production of the plasmid vector, molecular cloning procedures were performed. Cloning of APOE4 was performed by ligation of DNA fragments containing compatible cohesive ends and cloning of hSYN by ligation of fragments containing blunt ends. Twenty colonies of murine hepatocytes with the deleted BCR were obtained, 15 of which were monoallelic and 5 biallelic deletions. After PCR and restriction analysis, a bacterial colony was diagnosed as positive for APOE4 cloning. PCR genotyping allowed to diagnose two colonies as positive for hSYN cloning. In fact, the advances presented in this work puts us closer to the production of the new murine model for Alzheimer's Disease.