| Atrial fibrillation, long QT syndrome, and Middle East Respiratory Syndrome are three deadly human diseases for which genetic animal models are needed. From elucidating disease pathogenesis to facilitating the development of treatments, animal models are crucial for studying human disease. One of the most effective ways to generate specific animal models is through genetic modification. Historically, mice have been most widely used as genetically modified models, despite a number of limitations. New gene editing technologies such as CRISPR/Cas9 have made developing alternative genetic models that better recapitulate some human diseases better and more feasible. In this thesis, I describe my efforts to develop genetically modified goat and hamster models for atrial fibrillation and long QT syndrome, and genetically modified hamster models for Middle East Respiratory Syndrome. For long QT syndrome model development, I knocked out the KCNQ1 gene in goat fetal fibroblast cells and baby hamster kidney cells using the CRIPSR/Cas9 system. The knockout results in loss-of-function mutations, a known cause of human long QT syndrome. The edited goat fibroblast cells will be nuclear donors for future cloning experiments to produce live goats possessing the KCNQ1 knockout. The CRISPR gene targeting sgRNA/Cas9 vector, specific for the hamster KCNQ1, has been used for pronuclear injections to produce KCNQ1 knockout hamsters. For atrial fibrillation model development, I designed a single-stranded donor oligonucleotide that generates a KCNQ1 gain-of-function mutation resulting in the disease. This oligonucleotide was injected into hamster embryos along with the KCNQ1 sgRNA/Cas9-expressing vector to generate hamsters containing the gain-of-function mutation. Finally, for Middle East Respiratory Syndrome model development, I established a breeding colony of human DPP4 transgenic hamsters in the STAT2 knockout background. Human DPP4 transgenic hamsters are susceptible to MERS-CoV infection, showing mild clinical signs and allowing viral replication in lung tissue. Giving these hamsters a STAT2 knockout background should promote a more severe disease progression. For all three diseases, the foundations for the development of genetic animal models have been laid. |
