Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration
April 27th, 2018
This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.
The overall goal of this surgical procedure is to alter gene expression in the murine prostate by viral delivery of CRISPR Cas9 guides for prostate cancer induction. This method can help addressing new questions in prostate cancer biology regards to analyzing and validating new genes in an in vivo setting. The main advantages of this technique is it allow rapid gene editing of multiple genes by only targeting a subset of the prostatic cells as seen in the human scenario.
Someone who is new to the procedure may struggle at first because of organ size, and the precision that is needed for a successful surgical completion. To freshly prepare a total volume of 5 milliliters of anesthetic, mix 0.15 milliliters of one mg per milliliter medetomidine hydrochloride, 0.4 milliliters of 5 mg per milliliter of midazolam, and 0.25 milliliters of 10 mg per milliliter butorphenol. Then, add 4.2 milliliters of sterile saline water.
To reverse the effects of medetomidine hydrochloride post surgery, mix 0.1 milliliter of 5 mg per milliliter atipamezole with 4.9 milliliters of sterile saline water as an anesthesia antidote. Prepare an aseptic surgical environment by proper disinfection and sterilization and autoclave the surgical instruments. To deliver virus to the prostate, after anesthetizing an eight-week-old male mouse according to the text protocol;examine the anesthetic depth by assessing muscle relaxation, pedal withdrawal, and palpable reflexes.
When loss of reflexes is observed, shave the lower abdomen of the animal. Using a sterile cotton swab, carefully cover the animal's eyes with veterinary ophthalmic ointment to prevent blindness caused by xerophthalmia. Then, use 70%ethanol and 10%providone iodine to wipe the shaved abdomen to disinfect the surgical area.
Next, using sterile surgical scissors, make an approximately one centimeter vertical skin incision at the low abdominal midline. Then, with a fine point forceps, lift the peritoneum to prevent damaging the organs that are laying underneath;and use surgical scissors to carefully make an eight millimeter or shorter incision through the peritoneum. Gently move the fat tissue aside to uncover the seminal vesicle.
Then, using a ring forceps, carefully lift up the seminal vesicle until the anterior prostate can be identified. Now, with a 0.5 milliliter insulin syringe and a 30 gauge 8 millimeter needle;inject a total volume of 30 microliters of virus solution into the anterior prostate epithelium. Minimize leakage and ensure the fluid is absorbed within the tissue, forming a small bubble.
Then, place the seminal vesicle back in the abdominal cavity. To ensure the fluid is absorbed within the tissue in a small bubble and without leakage, make sure to inject parallel to the seminal vesicle and following the shape of the anterior prostate lobe. With a taper point needle and a 13 millimeter 3/8 circle, suture the perineum with two to three simple interrupted stitches of 6-0 absorbable suture.
Then, lifting the skin with forceps to avoid damaging the peritoneum;staple the skin with three sterile 4.8 by a 6.5 millimeter clips. For better recovery following surgery, use a sterile 1 milliliter syringe and a 27 gauge by 1/2 inch needle to administer the anesthesia antidote in a dose of 0.1 milliliters per gram body weight by intraperitoneal injection. Then, carefully place the animal back in its cage.
Keep the cage on a heating pad for one hour after the procedure to prevent hypothermia until fully recovered. The animal should awaken a few minutes after the injection. Continue to monitor and treat the animal daily according to the text protocol.
And, when the wound has closed, remove the skin clips. To assess virus delivery to the murine prostate, samples were analyzed three months after surgery. As shown here, the mice used in this study expressed GFP in cells that have been exposed to the Cree protein expressed by the virus.
The GFP signal shows Cree activity in the prostate epithelium but not whether gene editing had been induced by the CRISPR guide. These immunohistochemical sections identify focal areas of cells with high expressions of pAKT, indicating loss of P10. In these panels, costaining for pAKT and GFP identified double positive cells, which confirmed that transformation of prostatic cells by the adeno-associated virus.
Over all, these results show that in vivo, CRISPR-Cas9 gene editing can be performed in the prostate epithelium by using the Rosa26-LSL-Cas9-EGFP mouse and the adeno-associated virus. Once mastered, the procedure can be done in 15 minutes. This technique paved the way for research in the field of prostate cancer to examine multiple genes with simultaneous in vivo.
After watching this video, you should have a good understanding of how to deliver substrate to the murine prostate. Don't forget that when working with some viruses can be extremely hazardous and precautions should always be taken when doing these procedures.
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Overview
This protocol describes a method for virus delivery to the murine prostate, utilizing CRISPR/Cas9 technology for gene editing. This technique enables orthotopic alteration of gene expression and introduces a novel mouse model for studying prostate cancer.
Key Study Components
Area of Science
- Neuroscience
- Gene Editing
- Prostate Cancer Research
Background
- The surgical procedure aims to alter gene expression in the murine prostate.
- Viral delivery of CRISPR/Cas9 guides is employed for prostate cancer induction.
- This method addresses new questions in prostate cancer biology.
- It allows for the analysis and validation of new genes in an in vivo setting.
Purpose of Study
- To enable rapid gene editing of multiple genes.
- To target a subset of prostatic cells, mimicking human scenarios.
- To improve understanding of gene functions in prostate cancer.
Methods Used
- Preparation of anesthetic for surgical procedures.
- Viral delivery techniques for gene editing.
- CRISPR/Cas9 technology for precise gene alteration.
- Use of a novel mouse model for in vivo studies.
Main Results
- Successful alteration of gene expression in murine prostate.
- Demonstrated potential for rapid gene editing.
- Validated the effectiveness of the new mouse model.
- Provided insights into prostate cancer biology.
Conclusions
- The method offers a promising approach for prostate cancer research.
- It enhances the ability to study gene functions in vivo.
- Future studies can build on this technique to explore new therapeutic avenues.
Frequently Asked Questions
Executive Industry Relevance
This method enables targeted gene editing in a physiologically relevant prostate cancer model by delivering CRISPR guides via viral vectors to a subset of murine prostate epithelial cells. It addresses a key challenge in oncology R&D: modeling the clonal initiation and progression of tumors that arise from rare cellular events, as seen in human prostate cancer. By reducing reliance on germline models and extensive breeding, the approach accelerates target validation and mechanistic de-risking in discovery pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by inducing precise genetic alterations in a small subset of prostate epithelial cells, mimicking human tumor initiation.
- Operational Value: Supports rapid functional validation of candidate genes and pathways without requiring germline crosses or long latency periods.
- Predictive Value: Enhances confidence in target selection by modeling clonal expansion and early transformation events relevant to human disease.
Screening & Assay Development
- Scientific Value: Generates reproducible, in vivo-derived biological systems for assessing the oncogenic potential of genetic perturbations.
- Operational Value: Provides a standardized platform for quantifying phenotypic outcomes such as pAKT activation and GFP reporter expression in defined prostate lobes.
- Scalability Value: Enables parallel testing of multiple guide RNAs or viral constructs in cohorts to support lead identification efforts.
Translational & Preclinical Research
- Scientific Value: Models prostate cancer progression from initiation to invasion through basal membrane, supporting studies of microenvironmental interactions and metastatic potential.
- Operational Value: Allows longitudinal monitoring of tumor development in immunocompetent mice, improving translational fidelity for preclinical efficacy testing.
- Risk Mitigation: Reduces false positives in target validation by restricting genetic alterations to anatomically and biologically relevant cell subsets.
Pipeline & Workflow Integration
The technique fits within the discovery-to-preclinical continuum by enabling in vivo target validation, supporting assay-ready model generation, and informing go/no-go decisions based on phenotypic readouts such as transformation markers and pathway activation.
- Discovery Biology: Facilitates hypothesis-driven gene editing to assess oncogenic drivers and tumor suppressor loss in a spatially controlled manner.
- Assay Readiness: Produces quantifiable outputs including GFP reporter expression and immunohistochemical staining for pAKT, enabling objective comparison across experimental conditions.
- Analytics: Supports statistical analysis of transformation frequency and phenotypic penetrance in targeted cell populations, aiding in target prioritization.
- Translational Research: Models human-relevant tumor evolution from focal initiation to invasive progression, supporting biomarker co-localization studies.
- Enterprise Reuse: Establishes a reusable surgical and viral delivery platform applicable to multiple genes, pathways, and combination studies in prostate cancer research.
Operational & Enterprise Impact
- Scientific Value: Improves predictive confidence in target validation by reducing mechanistic ambiguity through spatially restricted, inducible gene editing.
- Operational Value: Standardizes tumor induction via reproducible surgical injection and viral titration, minimizing inter-animal variability.
- Strategic Value: Enables faster iteration on target hypotheses, reducing time and cost associated with traditional germline model generation.
- Portfolio Impact: Supports risk-adjusted advancement by providing early in vivo evidence of gene function in a clinically relevant anatomical context.
Implementation Considerations
- Requires expertise in microsurgical techniques, anesthesia management, and postoperative care for murine models.
- Dependent on access to biosafety-rated facilities for viral vector handling and sterile surgical instrumentation.
- Necessitates standardization of viral titer, injection volume, and anatomical targeting to ensure reproducible transduction efficiency.
- Requires validation of cell-type specificity and absence of off-target tissue transduction, particularly in urinary tract-adjacent regions.
- Limited by the payload capacity of viral vectors and potential immune responses to viral components or transgene expression.
