Applicable species and strains: mice C57BL/6J, BALB/c, CD-1 (6-8 weeks, 18-25 g); rats Sprague-Dawley, Wistar (6-8 weeks, 150-250 g) [12-1,12-8]. Dose range: mouse liver/heart/muscle targeting 1×10^11-1×10^12 vg/mouse (approximately 5×10^12-5×10^13 vg/kg), BBB-penetrating engineering capsid (PHP.B type) 1×10^11-1×10^12 vg/mouse; rat CNS targeting AAV9 3×10^13-1×10^14 vg/kg[12-1,12-7,12-8]. Injection volume: 100-300 μL for mice (the upper limit is about 10 mL/kg); 200-500 μL for rats [12-1]. Needles: 27-30 G insulin syringe for mice; 28-30 G catheter or 25 G needle for rats [12-1]. Speed: manual slow injection for 30-60 s, fast bolus injection is prohibited [12-1]. Anesthesia: Awake operation with heated fixator; PHP.B protocol polyisoflurane inhalation anesthesia [12-1]. Operating points: Expand the tail vein in a 37°C water bath before injection, insert the needle from the lateral/dorsal vein of the distal 1/3 of the tail (<15°), the bolus is successful if there is no resistance and the vein turns white, and the cotton ball is pressed for 10-30 s; the interval between multiple injections is ≥ 2 weeks, and repeated administration is limited by anti-capsid neutralizing antibodies [12-1, 12-12].
Coverage: liver (AAV8/9 is the strongest, 90%+ hepatocytes with dose), heart (AAV9 preferentially myocardium), skeletal muscle; AAV9 and engineered capsids can cross the BBB; expression peaks in 2-6 weeks and lasts for a long time [12-6]. Main off-target: liver is the largest off-target organ, followed by spleen, kidney, and lung; high-dose DRG neurons are enriched [12-11]. Limitations: Strong dependence on mouse strains (AAV-PHP.B is only effective in C57BL/6J lines and related to Ly6a) [12-7]; see section 8.4 for the risk of high-dose hepatotoxicity/complement activation.