DROSHA Knockout Leads to Enhancement of Viral Titers for Vectors Encoding miRNA-Adapted shRNAs.

ABSTRACT: RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNA^miR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNA^miR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packagin

SUBMITTER: Park HH 

PROVIDER: S-EPMC6078836 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature