Portal vein injection, as a novel tool to generate syngeneic orthotopic models of liver tumors that avoid most of the weaknesses of existing syngeneic models, has been developed. In this protocol, we here present the experimental procedure of portal vein injection in the mouse as follows. Preparation
●Before surgery, mouse is anesthetized by intraperitoneal injection of 2 % sodium pentobarbital (50 mg/kg). Wait until the mouse becomes motionless (~5 min).
●Place the mouse in a supine position, on its back with abdomen exposed. Remove the fur on the ventral left side of the rodent from the second rib space down to the 4th inguinal mammary gland nipple and then swab the region with 70 % ethanol repeatedly. This step can be completed 1 days in advance to save time when the mouse is awake.
Injection
●Make a 3-cm-long incision along the area from the second rib space down to the 4th inguinal mammary gland nipple with a sterilized scalpel.
Note: Be careful not to damage the breast, intestine, liver and other organs.
●Use a sterilized cotton swab to carefully pull out the internal organs and gently cover it with gauze to prevent touching until the hepatic portal vein is clearly visualized.
●Prepare a 32-gauge needle containing the solution to be injected. Slowly insert the needle 3-5 mm into hepatic portal vein 1cm below the liver at an angle of less than 5°.
Note: If necessary, a dissecting microscope may be used.
●Push on the syringe and slowly inject the solution, then retain the needle approximately 3-5 sec after finishing delivery. Remove the needle while simultaneously placing a sterile cotton swab on the vein with pressure. Hold the hemostatic gauze at the injection site with pressure from a sterile cotton tip applicator for 5 min.
Note: This step of hemostasis is vital for during the surgical procedure.
Post-injection care
●Remove the hemostatic cotton from the injection site. A small amount of sterile PBS can be used to soak if the hemostatic cotton sticks to the surrounding tissue. Place the internal organs gently back into the abdominal cavity and suture the incision with sterile 4-0 sutures. Typically, closing the incision requires 10-15 sutures.
●Inject 100 µl of bupivacaine (5 mg/ml) along the incision site for local pain management using an insulin syringe. Inject 0.5 ml of sterile PBS subcutaneously using a 1 ml syringe with 26-gauge needle for hydration. The whole surgeries take 15 - 25 min to complete.
●Place the mouse back to it’s cage next to the electric heater until it recovers.

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