Assessment of shRNA-mediated ATAD3 knockdown via lentiviral vectors in mouse adult fibroblasts

The aim of this thesis work was to assess and verify the silencing efficiency of short hairpin RNA constructs targeted against ATAD3A mRNA in mouse adult fibroblasts through infection with lentiviral vectors, following cell selection based on puromycin resistance. RNA interference-mediated protein silencing can be induced via shRNAs, RNA molecules that cause gene expression knockdown by degrading host mRNA, thus inhibiting specific protein synthesis. In this study, the target was ATAD3A mRNA (ATPase Family AAA Domain Containing 3A). The ATAD3A gene is part of a 3-gene cluster in hominids, but it is present as a single gene in the rest of pluricellular eukaryotes. ATAD3A is located in the inner mitochondrial membrane and regulates processes such as mitochondrial network organization, metabolism, and cell growth. Studies have shown that mutations in the ATAD3A gene have been associated with neurodevelopmental disorders. To investigate the role of ATAD3A at the molecular level, our lab designed a strategy to silence ATAD3A expression. To this aim, a design web tool was used to identify proper targeting sequences at the murine ATAD3A mRNA. A proper vector was chosen to insert the different shRNA sequences targeting the ATAD3A mRNA, then proceeding with virus production and cellular transduction. To evaluate the success of the approach, a combination of western blot and PCR techniques was applied. Moreover, the key role of puromycin as a selective agent added to cell cultures was investigated. Our findings provide an effective protocol to generate successful ATAD3A gene expression knockdown through shRNAs. This highly specific mechanism could help clarify the molecular function of ATAD3A, useful to understand mitochondrial diseases related to mutations in ATAD3A.