In Vivo Expression of Mutant Calpains in the Eye Using Lentivirus
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.
Key wordsCalpain CAPN5 Lentiviral vectors Molecular cloning Subretinal injection Intravitreal injection Mouse eye
VBM is supported by NIH grants [R01EY026682, R01EY024665, R01EY025225, R01EY024698, R21AG050437, and P30EY026877], The Doris Duke Charitable Foundation Grant #2013103, and Research to Prevent Blindness (RPB), New York, NY.
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