Block it adenoviral expression system

Recombinant adenoviruses encoding shRNAs to target rat JIP1 were prepared using the BLOCK-it™ Adenoviral Expression System (Invitrogen). Oligonucleotide targeting sequences are shown in Table 1. Top and bottom strands were annealed and ligated into the pENTR/U6 vector (Invitrogen). Positive subclones were recombined with the pAd/BLOCK-it™-DEST vector (Invitrogen) and then transfected into 293A cells using Fugene 6 (Roche). PCR was employed to construct full-length JIP1 (JIP1-FL, amino acids 1–707) and N-terminally truncated JIP1 (JIP1-ΔN, amino acids 94–707) expression vectors using mouse JIP1 as a template (Addgene plasmid #51699) [23 (link)]. JIP1-FL was cloned into pcDNA3.1 with an in-frame N-terminal FLAG epitope tag. JIP1-ΔN was cloned into pcDNA3.1 with an in-frame N-terminal Myc epitope tag. Next, both cDNAs were excised and subcloned into pENTR2b. LR-recombination was then performed to transfer the JIP1 cDNAs from pENTR2b to pAd-CMV-V5 DEST. Recombined viral vectors were transfected into 293A cells using polyethyleneimine (PEI). Viruses were amplified and recovered from 293A cells, and titered using the Sea-Plaque agarose method. All viruses were used at a multiplicity of infection (MOI) of 50 at the time of plating.