Fluorescent Orthotopic Mouse Model: Implanting GFP Expressing Cancer Cells into Mouse for Assessing Tumor Progression In Vivo

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the jove veterinary review board.

1. Surgical Implantation

NOTE: Ensure that all surgical materials and instruments are sterile. Practice aseptic techniques at all times.

1. Place a heating pad on the table and cover with a sterile drape. Set up the anesthesia machine and ensure all the supplies are within arm's reach.
2. Place the animal's snout into the anesthesia mask and set up the anesthesia to 1 L/min of oxygen and 2.5% isoflurane.
3. Gently scrub the flank of the animal with iodine, and rinse with 70% ethanol. Repeat three times.
4. Confirm the animal is fully anesthetized by pinching the hind paw. The animal is fully anesthetized and ready for surgery if no response is observed. However, if the animal flinches, ensure there is sufficient anesthesia in the vaporizer and allow the animal more time to go completely under anesthesia.
5. Load approximately 200 µl of the cell suspension into a 1.0 ml TB syringe with an 18 G needle (previously cooled). Replace the needle with a 27 G needle and return to ice until ready to use.
6. Locate the general area of the spleen (left upper quadrant of the abdomen) and using forceps pinch the skin on top of that region. Using surgical scissors make an incision of approximately 1.0 cm to create a pocket. Similarly, pinch the smooth muscle on top of the spleen and cut through in order to access the peritoneal cavity.
7. Gently grab the caudal end of the spleen and pull it out of the body. The pancreas will be attached to the spleen. Spread the pancreas using a wet sterile Q-tip and locate the tail of the pancreas.
8. Deliver the 50 µl injection into the tail of pancreas, leave the needle inside for 10 sec and slowly rotate the needle out of the pancreas. A successful implantation will look like a superficial bubble without any leaks.
9. Return the pancreas and spleen to the peritoneal cavity. First enclose the muscle and then enclose the skin separately. Use a 6-0 suture or staple to close the incision. To avoid pain in the animal, administer ketoprofen subcutaneously (SC) (5 mg/kg) over 24 hr. Alternatively, administer buprenorphine sc (0.05 - 0.1 mg/kg) every 12 hr over a 36 hr period. 
10. Recover the animal from anesthesia and return it to its cage. Also monitor for pain. Animals must be provided with pain relief at the time of (or even prior to — pre-emptive) surgery. Additional pain relief must be provided to animals that experience pain according to IACCUC-protocol or as prescribed by the attending veterinarian or designee.

자료

Name	Company	Catalog Number	Comments
Alcare or Foamed Antiseptic Handrub	Steris	639680
TB Syringe 27 G 1/2	Becton Dickinson	305620
Isoflurane	Blutler Schein	50562
Ketoprofen	Fort Dodge Animal Health
Surgical Scissors, 5.5" straight mayo	Henry Schein	22-1600
PANC-1 GFP cell line	Anticancer, Inc
Small Animal Imaging System: iBox Scientia	UVP, LLC. Upland, CA.		Small Animal Imaging System to observe the fluorescent tumor in live animals
SutureVet PGA 6-0 PGA	Henry Schein	39010
PANC-1 GFP cell line	Anticancer, Inc
DPBS (Dubelcco's Phosphate Buffered Saline)	Thermo Ficher Sci	21300025