Amicon ultra centrifugal filter 100 k

For lentivirus preparation for in vitro experiments, the second generation packaging system (which generates replication-deficient lentivirus) was used for all experiments (Zufferey et al., 1997 (link)). Packaging vectors psPAX2 and pMD2.G were obtained from Addgene. Briefly, lentiviral constructs for shRNA or overexpression were co-transfected with the packaging vectors into HEK293T cells with calcium phosphate. Supernatants containing virus were collected 24 h and 48 h after transfection. After centrifugation at 1000 rpm for 10 min, the supernatants were passed through a 0.45 μm PDVF filter unit (Nalgene). The viruses were concentrated 20–30x by centrifugation in an Amicon Ultra centrifugal filter (100 K) (Millipore) following the manufacturer's instructions. Viruses were aliquoted and stored at −80°C. Viral titers ranged from 1–5 × 10⁶ infectious units/μl. For in vivo experiments, virus preparations from above were further concentrated by ultracentrifuge at 50,000 rpm (Beckman TL-100 Ultracentrifuge) at 4°C for 1 h. Pellets containing virus were resuspended in PBS. Titers varied between 10⁷ and 10⁹ infectious units/μl.

To construct AAV2-EF1α-FLEX-eGFP-2a-TVA and AAV2-EF1α-FLEX-RG, TVA and eGFP linked by the 2A ‘self-cleaving’ peptide or rabies glycoprotein was respectively cloned into pAAV-MCS (Stratagene, La Jolla, CA) in an antisense direction flanked by a pair of canonical loxP sites and a pair of lox2272 sites. AAV particles (AAV2/2) were produced by co-transfection of packaging plasmids into HEK293T cells, and cell lysates were fractionated by iodixanol gradient ultracentrifugation. Viral particles were further purified from the crude fraction by heparin affinity column (HiTrap Heparin HP Columns; GE Healthcare, Pittsburgh, PA), desalted and concentrated with Amicon Ultra Centrifugal Filter (100 K, Millipore, Bellerica, MA). The genomic titer of AAV2-EF1α-FLEX-eGFP-2a-TVA (4.4 × 10¹³ gc/ml) and AAV2-EF1α-FLEX-RG (2.2 × 10¹² gc/ml) was estimated by quantitative PCR. eGFP-2a-TVA and rabies glycoprotein were subcloned from the AAV-TRE-HTG plasmid from L. Luo.
RV-ΔG-tdTomato was amplified in B7GG cells and pseudotyped using BHK-EnvA cells. EnvA pseudotyped rabies virus was titered (1.5 × 10⁹ IU/ml) by infecting the 293T-TVA8000 (Narayan et al., 2003 (link)) cell line with serial dilutions of the stock virus. RV-ΔG-tdTomato was a gift from B. Lim. B7GG cells, BHK-EnvA cells (Wickersham et al., 2007 (link)), and 293T-TVA8000 cells were gifts from E. Callaway.

The human pull of shRNA NSun2 and scramble shRNA control were obtained from Applied Biological Materials Inc. To prepare lentivirus for in vitro experiments, the second-generation packaging system (which generates replication-deficient lentivirus) was used for all experiments [99 (link)]. Packaging vectors such as psPAX2 and pMD2.G were obtained from Addgene. Briefly, lentiviral constructs for shRNA or scramble control were co-transfected with the packaging vectors into HEK293T cells using CalFectin (SignaGgen). Supernatants containing virus were collected 48 h after transfection. After centrifugation at 1000 rpm for 10 min, the supernatants were passed through a 0.45 μm PDVF filter unit (Nalgene). The viruses were concentrated 20–30 × by centrifugation in an Amicon Ultra centrifugal filter (100 K) (Millipore) following the manufacturer's instructions. Lentiviral stock titration was carried out using the Global UltraRapid Lentiviral Titer Kit (System Biosciences). Viruses were aliquoted and stored at − 80 °C.