AAV9 transduction of circ-ZNF609

For knockdown and overexpression of circ-ZNF609 and ZNF609-250aa in vivo, full-length sh-circ-ZNF609, ZNF609-250aa, or the control sequence was inserted into a pAV-U6-GFP vector. After the accuracy of the vector was measured by sequencing, AAV serotype 9 (AAV9) was packaged, purified, and titrated by Vigene Biosciences. One hundred microliters of AAV9 (5 × 10¹³ vg/mL) harboring either sh-circ-ZNF609 or ZNF609-250aa or the control sequence was injected into the renal veins of rats.⁴¹ Briefly, the rats were anesthetized; skin, muscle, and fascia were cut; the kidneys were exposed; and then the renal vein was clamped by a microaneurysm clamp. Finally, we injected the AAV particles mentioned above diluted in 100 μL of saline into the renal vein with a 31G needle and removed the clamp 15 min after injection, followed by suturing the incision.