Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells

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

1. Virus-delivery to the Murine Prostate

1. Anesthetize the animal by intraperitoneal injection with a sterile 1 mL syringe and a 27G x ½" needle. Use an anesthesia dose of 0.01 mL/g body weight. If the procedure lasts longer than 30 min, maintain anesthesia by injecting 1/3 of the starting dose every 30 min.
2. Examine the anesthetic depth by assessing muscle relaxation, pedal withdrawal, and palpebral reflexes. When loss of reflexes is observed, shave the lower abdomen of the animal.
3. Carefully cover the animal's eyes with veterinary ophthalmic ointment using a sterile cotton swab to prevent blindness due to lack of corneal reflex.
4. Wipe the shaved abdomen with 70% ethanol and 10% povidone-iodine to disinfect the surgical area. Scrub the surgical area from the center of the surgical area toward the periphery.
5. Perform a vertical skin incision at the low-abdominal midline of approximately 1 cm using a sterile surgical scissor.
6. Lift the peritoneum using fine point forceps to prevent damaging the organs that are lying underneath. Carefully make a <8 mm incision using surgical scissors through the peritoneum.
   NOTE: In contrast to humans, a rodent's prostate gland comprises four separate lobes. The anterior lobe is attached to the seminal vesicle.
7. Gently move the fat tissue aside to uncover the seminal vesicle. Using a ring forceps, carefully lift up the seminal vesicle until the anterior prostate can be identified (Figure 1).
8. Inject a total volume of 30 µL virus (3 x 10¹⁰ viral particles) solution in the anterior prostate epithelium using a 0.5 mL insulin syringe with a 30G x 8 mm needle. Minimize leakage and ensure the fluid is absorbed within the tissue, forming a small bubble. Place the seminal vesicle back in the abdominal cavity.
9. Suture the peritoneum with 2-3 simple interrupted stitches using 6-0 absorbable sutures with a 13 mm, 3/8 circle, and a tapered point needle.
10. Staple the skin with three sterile 4.8 x 6.5 mm clips lifting the skin with forceps to avoid damaging the peritoneum.
11. For better recovery, apply the anesthesia antidote in a dose of 0.01 mL/g body weight by intraperitoneal injection using a sterile 1 mL syringe and a 27G x ½" needle. Carefully place the animal back in its cage.