Paav camkiiα egfp

An AAV vector expressing eGFP under CaMKIIα promoter (pAAV-CaMKIIα-eGFP) was purchased from Addgene (#50469). Mouse Dnmt3a (GenBank: AF068625.2) was cloned from mouse brain cDNA, inserted into pAAV-CaMKIIα-eGFP using restriction enzyme EcoR I and Hind III, then sequenced by BGI (China). Viral particles were produced as described in the rAAV Production protocol of AAV- DJ/8 Helper Free Packaging System (Cell Biolabs, USA). pAAV-CaMKIIα-eGFP-Dnmt3a, pAAV-DJ/8^{61 (link)} and pHelper (Cell Biolabs, VPK-400-DJ-8) were co-transfected (1.5:1:1) into HEK293T at 70% confluence using the lipofectin-mediated transfection method (Lipofectamine™ LTX, Invitrogen). Cells were incubated for 72 hours at 37 °C with 5% CO₂. After 72 hours, cells were collected to purify virus using an AAV purification kit (Biomiga, USA). Purified AAV particles were titered for genome content using real-time qPCR (Promega, USA). Titers were 6 × 10¹² GC (Genome Copy) per milliliter. Control virus, rAAV-CaMKIIα-GFP, was obtained from HANBIO company (China), with a titer of 1.3 × 10¹² GC.

DNA plasmids encoding pAAV-CaMKIIα-hM3Dq-internal ribosomal entry site (IRES)-mCitrine (Addgene, plasmid #50466), pAAV-CaMKIIα-hM4Di-mCherry (Addgene, plasmid #50477), pAAV-CaMKIIα-hChR2(H134R)-EYFP (Addgene, plasmid #26969), pAAV-CaMKIIα-EGFP (Addgene, plasmid #50469), and pAAV-Ubi-eGFP (Addgene, plasmid #62518) were obtained from Addgene. pAAV-CaMKIIα-Cre-GFP was constructed from Cre-GFP empty vector (Addgene, plasmid #20781) into pAAV-CaMKIIα-hChR2-EYFP (Addgene, plasmid #26969). Plasmid DNA was amplified, purified, and collected using a standard plasmid maxiprep kit (Qiagen). The purified plasmids were mixed into CaCl₂ solution with the DNA plasmid coding AAV-DJ and co-transfected into HEK293T cells using calcium phosphate precipitation methods. Transfected cells were harvested at 72 h after transfection and the virus was purified using the AAV purification mega kit (Cell Biolabs, Inc.). Viral titers were 5 × 10¹² particles/ml and stored in aliquots at −80 °C until use.