Intracardiac Delivery of Viral Vectors: A Technique to Inject Viral Vectors Intracardially to Study Pancreatic Metastasis in Murine Model
筆記録
– Metastatic cancer cells spread from one site to different body areas via blood circulation or lymph system. To study metastatic factors, we introduce retrovirus containing an oncogene somatically into a transgenic mouse. This mouse model is genomically altered by silencing the tumor suppressor gene, allowing the oncogene to express in the cell. The mouse soon develops a pancreatic tumor, similar to human tumorigenesis.
Begin by injecting ketamine-xylazine solution intraperitoneally to anesthetize the transgenic mouse. Now, place the shaved mouse on a surgical bed, and secure the limbs with lab tape. Mark a location on the center of the sternum for injection.
Load the syringe with viral suspension and insert the needle into the marked location. A red pulse of blood in the syringe confirms the needle location in the left ventricle. Inject the viral suspension whenever the pulse of blood appears in the syringe and quickly remove the needle. Apply gentle pressure to reduce bleeding.
Now, transfer the mouse to a cage and let it recover. Within a few weeks of injection, the injected virus infects the pancreas and develops a tumor, which soon metastasizes into other organs. In the following protocol, we will perform an intracardiac injection into a murine mouse model.
– Begin the infection procedure by first anesthetizing a seven-week-old RIP-Tag; RIP-tva mouse. Apply eye lubricant to both eyes to prevent corneal drying. Shave the hair from the chest cavity of the RIP-Tag; RIP-tva mouse. Position the mouse on its back with the chest facing up on an absorbent lab bench paper on top of a SnuggleSafe warmer.
Perform a toe pinch to check the animal for non-responsiveness and to confirm full anesthesia effect. Extend the front limbs and secure them with tape. Mark the chest of the mouse for injection. Mark a location midway between the sternal notch and the top of the xiphoid process and slightly left anatomically of the sternum.
Then, scrub the anterior chest wall with either a povidone-iodine scrub or a chlorhexidine scrub, followed by a 70% isopropyl alcohol or a 70% ethanol-soaked gauze sponge. Draw 50 microliters of air into a sterile 28 and 1/2 gauge insulin syringe to create space between the plunger and the meniscus of a 500-microliter syringe. Draw up 100 microliters of virus.
The airspace without any liquid on the wall is crucial for seeing the cardiac pulse. Keep the needle upright. Hold the skin of mouse taut with one hand and insert the needle into the marked location.
When a bright red pulse of blood appears in the syringe, stop advancing the needle and fix the depth of the needle in the mouse with one hand. Use the other hand to carefully and slowly push the plunger to deliver the viral suspension over about a 60-second period. Deliver 10 to 20 microliters whenever a bright red pulse of blood appears in the syringe. The continuous entrance of red, oxygenated blood into the transparent needle hub indicates the proper positioning of the needle into the left ventricle.
After the last push of the plunger to deliver viral suspension and before seeing the next red pulse of blood, quickly retract the needle out of the chest cavity. Apply gentle pressure over the injection site for at least one minute to reduce the bleeding. Then, carefully move the mouse onto a heating pad or under a heat lamp until it is fully conscious.
