Phelper

Adeno-associated viruses were produced at the Vector Facility of the University Medical Center Hamburg-Eppendorf (UKE). pAAV-hSyn1-mRuby2 (Addgene 99126; deposited by Viviana Gradinaru) and pAAV-hSyn1-GFP-synaptopodin (this study) were respectively packaged by pE2/rh10 and p5E/9 (Julie C. Johnston, University of Pennsylvania, USA) and pHelper (CellBiolabs). For infection with AAV, the viruses were added directly into the culture medium at final concentrations of between 10⁹ and 10¹¹ vg/ml. pSyn-GFP-Synaptopodin AAV9 was added to the culture at DIV10, mRuby2 AAVrh10 was added on DIV14, and cells were imaged on DIV17.

We used the AAV2 serotype to transfer sgRNAs into HeLa cells. AAV2 particles were packaged using the triple transfection method. First, a transfection mixture consisting of 14 µg pHelper (Cell Biolabs), 8 µg pAAV-RC2 (Cell Biolabs), 8 µg PX601-mCherry, and 60 µg Polyethylenimine (PEI) (sigma, 25 kDa, 1 mg/ml in H₂O, pH 7) was prepared in cell culture medium and incubated at room temperature for 15 min. Then, the mixture was drop-wised on 70–80% confluent HEK293t cells cultured in 10-cm plates. One day after transfection, the culture medium on the cells was replaced with a fresh medium (high glucose DMEM containing 2% FBS). Three days after transfection, the cells were harvested to release viral particles in three successive cycles of cooling on dry ice and heating in a 37° C water bath. Cell debris was collected by centrifugation at 10,000×g for 10 min at room temperature. AAV-containing lysates were treated with DNaseI for 30 min at 37 °C. The viral particles were then concentrated using 15 mL of 100 kDa molecular weight cut-off filters^{24 (link)}. AAVs were titrated using the quantitative polymerase chain reaction (qPCR) by ITR forward primer 5'-GGAACCCCTAGTGATGGAGTT-3’ and reverse primer 5'-CGGCCTCAGTGAGCGA-3’, according to Aurnhammer et al. protocol^{25 (link)}.

AAV was prepared as described previously (McClure et al., 2011 (link); Park et al., 2016 (link)). Briefly, three plasmids encoding the required components for AAV production were transfected into 293 AAV cells (Cell Biolab, Inc) using the calcium phosphate methods: AAV-DJ, pHelper (Cell biolabs, Inc), and pAAV-CaM kinase II. For production of MLI-specific AAVs (pX), a putative promoter region of pAAV-synapsin vector was replaced into promoter region of Nos-1, Grin3a, Kit and obtained pX-GFP and pX-cre AAVs using with Gateway system (Thermo Fisher Scientific). After 48–60 hr post-transfection, cells were solubilized and AAVs were purified using a HiTrap Heparin column (GE healthcare).

Packaging of plasmid AAV_Syn_TrkB.T1 or control plasmid pAAV_Syn_EGFP DNA into AAV capsids was performed by cotransfection of HEK-293FT cells with plasmids AAV-DJ (scAAV-DJ expression system by Cell Biolabs, Inc., San Diego, CA, USA) and pHelper (Cell Biolabs, Inc., USA) [23 (link)]. Harvesting of viral particles was performed in 48 h, in accordance with the protocol described by Grimm et al. [24 (link)]. The amount of the obtained viral particles was determined by real-time quantitative PCR (with primers 5′-cctggttgctgtctctttatgagg-3′ [forward] and 5′-tgacaggtggtggcaatgc-3′ [reverse]). A series of dilutions of an original plasmid of known concentration served as standards for determining the number of viral particles. Both AAV vectors utilized in this study had similar genomic titers (10⁹ viral genomes per microliter).

A luciferase reporter driven by a tandem repeat of the Gpr19 CRE sequence (3 × CRE-Luc2P) was inserted between the ITR sequences of pAAV-MCS vector (Cell Biolabs Inc) to obtain pAAV-3 × CRE-Luc2P. HEK293T cells cultured in dish were co-transfected with pAAV-3 × CRE-Luc2P, pAAV-DJ, and pHelper according to the manufacturer's instructions (Cell Biolabs Inc). Three days after transfection, cells were harvested and resuspended in 1 ml of DMEM, followed by four freeze–thaw cycles and centrifugation. The titers of 3 × CREwt-Luc2P and 3 × CREmut-Luc2P virus solutions were ~ 8 × 10¹² genome copies/mL. The SCN slices were prepared according to our standard method^{59 (link)}. Two days after the preparation of SCN slices, the AAV solution (3 μL per slice) was inoculated on the surface of the SCN slices. Infected slices were further cultured for ~ 14 days. Thereafter, luminescence from the culture was measured with a dish-type luminometer (Kronos Dio, ATTO) at 35 °C using 1 mM luciferin^{59 (link)}. The luminescence was monitored for 2 min at 20-min intervals for each slice. The raw data were smoothed using a 1-h moving average and further detrended by subtracting a 24 h running average.