Title | In Vivo Expression of Mutant Calpains in the Eye Using Lentivirus. |
Publication Type | Journal Article |
Year of Publication | 2019 |
Authors | Wert, Katherine J., and Mahajan Vinit B. |
Journal | Methods Mol Biol |
Volume | 1915 |
Pagination | 233-247 |
Date Published | 2019 |
ISSN | 1940-6029 |
Abstract | Exome sequencing has identified many candidate genes and mutations for human diseases, but the functional validation of these candidates is a time-consuming and costly process. Here, we describe a method which uses lentiviruses to overexpress calpain mutations that may play a role in dominant diseases such as autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). The use of lentivirus to deliver the mutant calpain allows for a cost-effective, rapid, and efficient approach to test whether or not a candidate gene mutation from exome sequencing acts as the disease-causing allele for a human disorder. This method also provides for a comparison of different candidate mutations from a single gene identified by exome sequencing, as well as elucidating the mechanisms underlying these complex human disorders. Furthermore, this chapter focuses on two different methods to deliver mutant calpain to the cells of the eye, using either a subretinal or an intravitreal injection of the lentivirus into the mouse eye. |
DOI | 10.1007/978-1-4939-8988-1_18 |
Alternate Journal | Methods Mol. Biol. |
PubMed ID | 30617808 |
Grant List | R01 EY026682 / EY / NEI NIH HHS / United States R01 EY024665 / EY / NEI NIH HHS / United States R01 EY025225 / EY / NEI NIH HHS / United States R01 EY024698 / EY / NEI NIH HHS / United States R21 AG050437 / AG / NIA NIH HHS / United States P30 EY026877 / EY / NEI NIH HHS / United States |