Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells
Published: April 30, 2023
Abstract
Source: Riedel, M. et al. Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration. J. Vis. Exp. (2018)
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. <…
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.
Divulgaciones
The authors have nothing to disclose.
Materials
| B6J.129(B6N)-Gt(ROSA)26Sor^{tm1(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 |
Citar este artículo
Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells. J. Vis. Exp. (Pending Publication), e20407, doi: (2023).