AAV9-STAT3 was obtained from WZ Biosciences Inc. as previously described [13 (link)]. Briefly, plasmid with the cytomegalovirus (CMV) promoter directing expression of human STAT3 was used to product AAV9-STAT3. And then, AAV9 vector was packaged in HEK293 cells by the double or triple transfection method. Ammonium sulfate fractionation and iodixanol gradient centrifugation were used to purify the virus. In the end, titers of the AAV9 vector were determined by quantitative real-time PCR (qRT-PCR) [14 (link)]. The green fluorescent protein- (GFP-) expressing vector (AAV9-GFP) was used as control.
In vivo, 60-80 μl AAV9-STAT3 (5.0-6.5 × 1013 genome copies(GC)/ml) was injected into the retroorbital venous plexus of the mice to overexpress STAT3 [15 (link)]. After one week, the mice underwent AB surgery or CTS administration [13 (link)]. Four weeks after AB surgery or CTS administration, mouse hearts were excised and total genomic DNA was extracted. qRT-PCR was used to detected AAV9 vector genome copy numbers.
In vivo, 60-80 μl AAV9-STAT3 (5.0-6.5 × 1013 genome copies(GC)/ml) was injected into the retroorbital venous plexus of the mice to overexpress STAT3 [15 (link)]. After one week, the mice underwent AB surgery or CTS administration [13 (link)]. Four weeks after AB surgery or CTS administration, mouse hearts were excised and total genomic DNA was extracted. qRT-PCR was used to detected AAV9 vector genome copy numbers.
