The overall goal of this procedure is to identify and characterize metastatic factors by gene transfer into the Novel RIP-Tag;RIP-tva mouse model. This method can help answer key questions in the cancer metastasis field such as identifying the drivers and the Novel therapeutic targets. The main advantage of this mouse model of cancer is to identify metastatic factors by somatic gene transfer into pancreatic beta cell precursor legions.
Begin by harvesting one 15 centimeter dish of virus producing DF1 cells per mouse to be infected. First, collect the medium from the confluent 15 centimeter dishes. Then remove cell debris by low speed centrifugation in a pre-chilled centrifuge for 10 minutes at 1650 times G and four degrees celsius.
Next, transfer the viral supernatant to polyallomer ultracentrifuge tubes. Spin in an ultracentrifuge for 1.5 hours at 9540 times G and four degrees celsius. Remove as much supernatant from the ultracentrifuge tubes as possible.
Then add PBS to the ultracentrifuge tube to make the final 100 microliter viral suspension per 15 centimeter dish. Cover the tubes with laboratory film and re-suspend the invisible viral pellet by vortexing for two minutes at medium speed. Then rock the ultracentrifuge tubes at four degrees celsius for one hour or overnight.
After the incubation, transfer the viral suspension to a microcentrifuge tube and proceed to titer the virus. Begin the titering procedure by ensuring the DF1 cells seated into 12 well tissue culture plates have reached 30%confluency. One twelve well plate is needed for each viral titer determination.
Begin a tenfold serial dilution of the viral supernatants by first mixing five microliters of viral supernatant with 495 microliters of growth medium in a microcentrifuge tube to make a ten to the second dilution. Vortex briefly for a few seconds. Then transfer 40 microliters of the ten to the second dilution to 360 microliters of growth medium in a microcentrifuge tube for the ten to the third dilution.
Vortex briefly for a few seconds. Continue this process to dilute to ten to the fourth and ten to the fifth. Next aliquot 2.25 milliliters of growth medium into each of five six milliliter polystyrene tubes.
And label them ten to the sixth, ten to the seventh, ten to the eighth, ten to the ninth, and ten to the tenth respectively. Mix 0.25 milliliters of the ten to the fifth dilution with 2.25 milliliters of growth medium in the six milliliter polystyrene tube for the ten to the sixth dilution. Vortex briefly for a few seconds.
Repeat the procedure for ten to the seventh, ten to the eighth, ten to the ninth, and ten to the tenth dilutions. Next remove the original culture medium from the DF1 cells on the 12 well plates. And add one milliliter of diluted virus into each well in duplicate.
Allow the cells to grow at 37 degrees celsius and 5 CO2 for at least seven days. 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 snuggle safe warmer. Perform a toe pinch to check the animal for nonresponsiveness and to confirm full anesthesia effect. Extend the front limbs and secure them with tape.
Mark the chest of the mouse for injection. Mark the 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 28.5 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 10-20 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 plunder 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. RIP-Tag RIP-tva mice were infected with the indicated RCASBP-Bcl-xL retro virus at seven weeks of age and euthanized at 16 weeks of age.
This photograph shows representative synaptophysin staining of metastatic pancreatic neuroendocrine tumors in pancreatic lymph nodes. Here the RIP-Tag RIP-tva mice were infected with the indicated RCASBP-RHAM B.This photograph shows the representative synaptophysin staining of metastatic pancreatic neuroendocrine tumors in pancreatic lymph nodes. Here the synaptophysin staining is seen in the liver after injection of the RCASBP-RHAMM B virus.
It is important to remember the title of greater than ten to the eight infectious units per milliliter is required for in vivo infection of proliferating TVA cells in RIP-Tag RIP-tva mouse and RCASBP retro viral rector can deliver CDNA up to 2.5 kilobases. After the development of this mouse model, it paved the way to a paradigm shifting finding that BCL-xL actually promotes cancer metastasis independent of it's antiapoptotic function.
