Overview
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.
Protocol
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
- 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.
- 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.
- Carefully cover the animal's eyes with veterinary ophthalmic ointment using a sterile cotton swab to prevent blindness due to lack of corneal reflex.
- 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.
- Perform a vertical skin incision at the low-abdominal midline of approximately 1 cm using a sterile surgical scissor.
- 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. - 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).
- 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.
- 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.
- Staple the skin with three sterile 4.8 x 6.5 mm clips lifting the skin with forceps to avoid damaging the peritoneum.
- 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.
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Representative Results
Figure 1: Illustration of the procedure. The procedure is carried out in the Rosa26-LSL-Cas9-EGFP mouse generated by Platt et al. The anterior prostate (AP) is attached to the seminal vesicle (SV). Virus particles expressing guide RNA and a Cre protein are injected into the anterior prostate to alter gene expression.
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Materials
| Name | Company | Catalog Number | Comments |
| B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J mice | The Jackson Laboratory | 26175 | |
| 0,5 ml U-100 insulin syringe | BD | 324825 | for virus injection |
| 1 ml syringe | BD | 300013 | for anesthesia injection |
| 30 G 1/2'' needle | BD | 304000 | for anesthesia injection |
| 6-0 Polysorb Suture | Medtronic | GL889 | to close the peritoneum |
| Disposable sterilized surgery drape | multiple suppliers | ||
| Heating pad | multiple suppliers | ||
| Povidone-Iodine Prep Pad | Fisher Scientific | 06-669-70 | |
| Trimming machine | Aesculap | GT415 | to shave the abdomen |
| Dumont Forceps | F.S.T | 11252-00 | |
| Halsey Micro Needle Holder | F.S.T | 12500-12 | |
| Iris Scissors | F.S.T | 14094-11 | |
| Narrow Pattern Forceps | F.S.T | 11002-12 | |
| Ring Forceps | F.S.T | 11106-09 | |
| Wound Clip System Handle incl. Clips |
F.S.T | 12030-01 | to close the skin |
| Wound Clip System Remover | F.S.T | 12030-04 | |
| 1x PBS | Gibco | 10010-023 | |
| Antisedan | obtained from the animal facility | ||
| Butorphanol | obtained from the animal facility | ||
| Medetomidinhydrochlorid | obtained from the animal facility | ||
| Midazolam | obtained from the animal facility | ||
| 100% Ethanol | Fisher Scientific | 22-032-103 | |
| Eye ointment | Takeda | 7242 | |
| Virus (AAV, AV) | multiple suppliers | virus used in this protocol is an in-house production |
