Lentivirus and Sindbis virus production

For lentivirus production, shRNAs driven by a H1 promoter were generated for the knockdown of rat SNX27 (target sequence 5’-aagaacagcaccacagaccaa-3’) (Binda et al., 2019), human SNX27 (target sequence 5’- aagaacagtactacagaccaa-3’), rat LRFN2 (target sequence 5’-acgacgaggtactgattta-3’), and a control (non-targeting sequence 5’-aattctccgaacgtgtcac-3’). Oligonucleotides were cloned into a modified pXLG3-GFP viral vector and co-transfected into a 15 cm dish of HEK293T cells with the helper plasmids Pax2/p8.91 and pMDG2 using PEI. For primary culture, the viruses were harvested 72 hr after transfection, spun down at 4000 rpm for 10 min at room temperature (RT) and filtered through 0.45 μm filters before being stored at −70°C. Neurons were transduced with shRNA viruses on DIV12 and left for 7 days before analysis. Only those experiments where knockdown resulted in more than 85% reduction of the protein of interest were used for analysis. For in vivo injections, the lentiviral constructs were modified to include a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to increase the GFP expression and the amount of HEK293T cells were scaled up to 10 x 15 cm dishes/virus. The media was harvested 48 hr after transfection, spun down at 4000 rpm for 10 min at RT and filtered through 0.45 μm filters. The filtered supernatant was then centrifuged in JA20 tubes at 6000 rpm overnight (O/N) at 4°C (Avanti J-25, Beckman Coulter). The following day the viral pellet was resuspended in 5 ml PBS and centrifuged at 20,000 rpm for 90 min at 4°C (Optima XL-100K, Beckman Coulter). The pellet was then resuspended in the required volume of PBS before being aliquoted and stored at −70°C.

For sindbis virus production, full-length human SNX27 and LRFN2 were cloned into the pSinRep5 plasmid. GFP-SNX27 was amplified from pEGFP-C1 and the resulting PCR product cloned into pSinRep5. The LRFN2 was subcloned from a pmCherryC1 vector which contained a mCherry fluorescent tag immediately after a N -terminal signal peptide (LRFN1 signal peptide) followed by full-length LRFN2. GFP- and mCherry- expressing pSinRep5 plasmids were created as controls. Five μg of in vitro-transcribed RNA (2.5 μg of SNX27/LRFN2 RNA and 2.5 μg of the defective helper plasmid) was electroporated into 0.6x10⁷ BHK-21 cells using a Gene Pulser II electroporation system (BioRad) in a gene pulser cuvette (0.2 cm gap). The electroporation conditions were set as follows: voltage; 1.5 kV, capacitance; 25 µF for a period of 0.7–0.8 ms. The viruses were harvested 36–48 hr after electroporation before being stored at −70°C. Neurons were transduced with sindbis viruses on DIV19/20 and left for 18–24 hr before analysis.