Packaging plasmids

A lentivirus transfer vector, based on a third-generation self-inactivating transfer vector (de Almeida et al. 2001 (link)), was designed to over-express a synaptophysin-mCherry fusion protein. The vector uses the phosphoglycerate kinase-1 promoter which expresses well in rat brain and is primarily neuronal (Krause et al. 2011 (link); Grillo et al. 2015 (link); Myers et al. 2017 (link)). Both rat synaptophysin (Open Biosystems MRN1768–99237971, clone 7936715) and mCherry (Clontech pmCherry-1) cDNAs were amplified by PCR, using primers designed to substitute GTG for the stop codon of the synaptophysin cDNA immediately upstream of the mCherry ATG. The two PCR fragments were cloned into the lentivirus transfer vector using Clontech Advantage HD Polymerase (Mountain View, CA). Viruses were generated by transfection of the transfer vector with three packaging plasmids (Addgene, Cambridge, MA), psPAX2, pRSV-Rev and pMD2.G, into 293T cells. Viruses were concentrated by high-speed centrifugation, purified by further centrifugation through 20% sucrose/Dulbecco’s phosphate buffered saline (DPBS) and stored in 10% sucrose/DPBS at −80°. Virus particle concentrations were determined by quantitative real-time PCR for proviral DNA 24 h following transduction of 293T cells and are expressed as transducing units per microliter (tu/μl).

Lentivirus was produced at titers >10E9 using methods similar to those described by Han et al. ^{11 (link)}. In short, 293T cells (Lenti-X Invitrogen 632180, Life Technologies) were transfected with the Lenti-backbone plasmid and packaging plasmids (Addgene, Cambridge, MA, USA). Supernatant was replaced after 24 h with Ultraculture medium (Invitrogen). Lentivirus was harvested by collecting the supernatant 48 and 72 h after transfection and spinning it at 22000 r.p.m (Beckman S28 rotor) through a 20% sucrose cushion in phosphate-buffered saline (PBS). Virus pellet was suspended by incubation in ice-cold PBS for 1-h, carefully mixed, aliquoted and frozen on dry ice for storage at −80°C. Titers were determined by preparing genomic DNA from 293T cells transduced with the Lentivirus and using quantitative PCR to compare amplification of a 150 bp lentivirus fragment with amplification of a comparable fragment of the endogenous human vasopressin receptor gene (hVAR1).

Lentivirus was produced at titers >10E9 as described by Han et al. 2010 ^{34 (link)}. In short, 293T cells (Lenti-X Invitrogen 632180) were transfected with the Lenti-backbone plasmid and packaging plasmids (Addgene). Supernatant was replaced by new medium after 24 hours with Ultraculture medium (Invitrogen ). Lentivirus was harvested by collecting the supernatant 48 hours and 72 hours after transfections and spinning it at 22,000 rpm (Beckman S28 rotor) trough a 20% sucrose cushion in PBS. Virus pellet was suspended by incubation in ice cold PBS for one hour, carefully mixed, aliquoted and frozen on dry ice for storage at −80C until needed for surgery. Titers were determined by preparing genomic DNA from 293T cells transduced with the Lentivirus and using qPCR to compare amplification of a 150 bp Lentivirus fragment with amplification of a comparable fragment of the endogenous human Vasopressin receptor gene (hVAR1). AAV vectors for the AAV serotype survey were stock vectors from the PENN vector core. AAV2-GluCla and AAV2-GluClb vectors ^{32 (link)} were produced by the UNC vector core at titers of 10E12 unites/ml. Viruses were shipped in aliquots on dry ice and stored at −80C until needed for surgery.