Lentivirus-Mediated NeuroD1 Overexpression and Knockdown

A Lentivirus V5-DEST construct expressing NeuroD1-cDNA was prepared as described previously [27 (link)]. The TRC Lentiviral shRNA of NeuroD (5’-CCGGGCTCAGCATCAATGGCAACTTCTCGAGAAGTTGCCATTGATGCTGAGCTTTTTG-3’) and a corresponding negative control construct were obtained from University of Minnesota Genomics Center. Virus particles were assembled in 293FT cells by transfecting V5-DEST constructs or shRNA constructs together with pLP1, pLP2 and pLP-VSVG constructs from the BLOCK-iT™ Lentiviral RNAi Expression System (Invitrogen, Grand Island, NY) following the manufacturer's instructions. Viral titers (~1.0 × 10⁸ TU/mL) were determined by assessment of DNA sequences in transduced 293FT cells [28 (link)]. NeuroD1 gene expression and knock down were tested by western blot analysis.