Amicon centrifugal lter unit

Lentivirus preparation, the construct was co-transfected to HEK293T cells pMD2g and psPAX. The original medium was changed with the fresh one. 48 hours after that, the medium was collected. The lentivirus particles were concentrated by centrifuging at 25,000 rpm for 2 hours. The production of lentiviral particles up to 109 IU/ml was puri ed by Ultra-Pure Lentivirus puri cation kits (Applied Biological Materials Inc. Vancouver). AAV preparation, the constructs were packaged into AAV9 by the MIRCen viral production platform. Brie y, viral particles were produced by transient co-transfection of HEK-293T cells with an adenovirus helper plasmid, an AAV packaging plasmid carrying the rep2 and cap9 genes, and the AAV2 transfer vector containing the above-mentioned expression cassettes by PEI. 72 h following transfection, virions were puri ed and concentrated from cell lysate and supernatant by ultracentrifugation on an iodixanol density gradient followed by buffer exchange to PBS, 0.01 % Pluronic via a 100kd Amicon Centrifugal lter unit (Merck-Millipore, Darmstadt, Germany). Concentration of the vector stocks was estimated by quantitative PCR and expressed as viral genomes per ml of concentrated stocks (vg/ml).

Lentivirus preparation, the construct was co-transfected to HEK293T cells pMD2g and psPAX. The original medium was changed with the fresh one. 48 hours after that, the medium was collected. The lentivirus particles were concentrated by centrifuging at 25,000 rpm for 2 hours. The production of lentiviral particles up to 109 IU/ml was puri ed by Ultra-Pure Lentivirus puri cation kits (Applied Biological Materials Inc. Vancouver). AAV preparation, the constructs were packaged into AAV9 by the MIRCen viral production platform. Brie y, viral particles were produced by transient co-transfection of HEK-293T cells with an adenovirus helper plasmid, an AAV packaging plasmid carrying the rep2 and cap9 genes, and the AAV2 transfer vector containing the above-mentioned expression cassettes by PEI. 72 h following transfection, virions were puri ed and concentrated from cell lysate and supernatant by ultracentrifugation on an iodixanol density gradient followed by buffer exchange to PBS, 0.01 % Pluronic via a 100kd Amicon Centrifugal lter unit (Merck-Millipore, Darmstadt, Germany). Concentration of the vector stocks was estimated by quantitative PCR and expressed as viral genomes per ml of concentrated stocks (vg/ml).