Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA

Our research focus is to enhance understanding of complex disorder progression using computational and experimental methods. We focus on developing strategy to slow down or reverse the progression of complex diseases such as metabolic disorders, aging, and cancer. The key question we're trying to address here is why some precancerous lesion develop into cancer, why some adult tissue can achieve regeneration after injury, and what's driving the aging process. These technologies include whole genome sequencing, single cell sequencing, robust computational and machine learning algorithm, AI modeling, and experimental animal models that are relevant to human diseases. This protocol describe a method for In-Vivo Gene Knockdown using intrasplenic injections of AAV8, delivered short hairpin RNA construct targeting nuclear stemming. Nuclear stemming is a stem cell self-renewal factors that plays important role in embryonic development, cancer development, and adult tissue regenerations. We showed that in transplanting injections of AAV8 deliver short hairpin RNA achieve the same knockdown efficiency. A set of portal vein injections of AAV8 deliver construct, but it's associated with much less complication and the procedure is much simpler.

[Instructor] To begin transfer the anesthetized mice to the aseptic surgical area in a right lateral recumbent position. Using a surgical blade, make a 15 millimeter incision on the left upper abdominal wall, followed by a 10 millimeter incision on the peritoneum. Using forceps, gently expose the spleen and place a piece of wet gauze underneath it to hold it in place. Next, using a 30 gauge needle, inject 50 microliters of AAV8 viruses into the spleen at a depth of three millimeters below the surface over a 30 second period. Then cover the site with a cotton swab and compress it for one minute. Afterward, suture the abdominal wall using a 4-0 absorbable suture. Then close the skin using a 4-0 non-absorbable suture. After anesthetizing, the mice perform a laparotomy by making a midline incision on the skin and then into the peritoneum. Place a sterile gauze pad soaked in sterile saline on the left side of the mouse. Using a sterile cotton swab, gently pull out the large and small intestines. Place the intestines on top of a wet gauze pad and cover them to prevent drying or contact with the skin. Then using a 32 gauge sterile needle, inject 50 microliters of AAV8 virus into the portal vein at an angle of less than five degrees. Insert the needle three to five millimeters into the portal vein and allow blood to flow past the needle for five seconds to prevent backflow. Next, place a sterile cotton swab on the injection site and apply gentle pressure until the vein is fully closed and no bleeding occurs. Afterward, gently return the internal organs back into the abdominal cavity. Then, close the abdominal wall using a 4-0 absorbable suture and the skin with 4-0 non-absorbable suture. Intrasplenic and portal vein injections showed statistically equivalent knocked down efficiency of the mRNA with no significant differences observed between the two methods.