Rxr γ sirna

Cav-1 siRNA, RXR-γ siRNA, and negative control (N.C.) siRNA were synthesized by Ribo Bio, Co., Ltd (Guangdong, China). A total of 100 μg siRNA was injected into mice via tail vein every 3 days for 5 injections (20 μg per injection). Endothelial-specific adeno-associated virus (AAV) was conducted by GeneChem co., Ltd (Shanghai, China). The AAV vector with Tie1 promoter (AAV-Tie1-MCS-EGFP-3Flag-SV40 PolyA) was employed to induce the expression of Cav1 in the endothelium. Briefly, the mouse Cav1 (NM_007616) was constructed on the AAV vector to generate the recombinant plasmid. AAV-293 cells were transfected with the recombinant plasmid. Three days after transfection, the recombinant AAV9 virus was assembled in the packaged cells. Then the AAV-293 cells were lysed to collect the virus supernatant, followed by CsCl density gradient centrifugation and ultrafiltration. Finally, viral titers were verified using quantitative PCR. The obtained Cav1-overexpressing AAV was named AAV-Tie1-Cav-1. The viral vectors were stereotaxically injected into the lateral ventricles (0.5 mm anterior–posterior, 1.0 mm medial-lateral, −2.0 mm dorsal-ventral relative to bregma) 3 weeks before tMCAO surgery. All mice received 3 μl of either AAV-Tie1-C (1.13 × E13 v.g/ml) or AAV-Tie1-Cav-1 (1.95 × E13 v.g/ ml).