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

Begin with an anesthetized and prepped CRISPR/Cas9 knock-in mouse model bearing a genome engineered to express Cas9 endonucleases and green fluorescent proteins, or GFPs, by a strong upstream promoter. A floxed-STOP cassette or LSL inserted immediately downstream to the promoter blocks Cas9 and GFP transcription under normal conditions.

Incise the mouse's abdominal wall to expose its anterior prostate lobe attached to the seminal vesicle. Inject the desired adenoviral suspension into the anterior lobe to facilitate targeted viral genome delivery into the cells. Place the prostate lobe back into the abdominal cavity and suture the incision.

Within virus-infected cells, the viral genome expresses Cre recombinase enzymes and guide-RNAs targeting the gene to be mutated. The Cre enzymes recognize the LSL cassette and excise the floxed transcription blocker. This step allows the promoter to drive the expression of Cas9 endonucleases and GFPs.

Subsequently, Cas9 endonucleases form complexes with virus-encoded guide-RNAs that direct these complexes to the target sequence within the gene to be mutated. This localization allows the Cas9 endonuclease to cleave the genomic DNA within the target gene, leading to genetic alteration.

An oncogenic alteration causes mutated cells to turn cancerous. The co-expression of GFP reporter proteins within mutated cells facilitates the identification and tracking of cancer progression.

To deliver virus to the prostate, after anesthetizing an 8-week-old male mouse according to the text protocol, 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. 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% povidone-iodine to wipe the shaved abdomen to disinfect the surgical area.

Next, using sterile surgical scissors, make an approximately 1 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 8 millimeter or shorter incision through the peritoneum. Gently move the fat tissue aside to uncover the seminal vesicle. Then, using a ringed forceps, carefully lift up the seminal vesicle until the anterior prostate can be identified.

Now, with a 0.5 milliliter insulin syringe and a 30G x 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 peritoneum 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 x 6.5 millimeter clips.

For better recovery following surgery, use a sterile 1 milliliter syringe and a 27G x 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.

• CRISPR/Cas9 knock-in mouse model - Genetically engineered rodent model used for research purposes involving the CRISPR/Cas9 genome editing system.
• Lsl cassette - A genetic construct used to block transcription under certain conditions.
• Cas9 endonucleases - Enzymes expressed by the Cas9 gene, used in CRISPR for cutting DNA at desired locations.
• Green Fluorescent Proteins (GFPs) - Proteins that glow green under specific light; used as a marker in biological research.
• Prostate Cancer Model - A mouse model designed for prostate cancer studies.

• Introduce CRISPR/Cas9 knock-in mouse model – Define and contextualize the genetically engineered rodent model utilized for genome editing research (e.g., mouse models of prostate cancer).
• Key Concepts – Summarize principles of genetic editing using concepts like lsl-cassette, Cas9 endonucleases, and GFPs (e.g., targeted viral genome delivery).
• Underlying Mechanisms – Briefly describe the process of creating a genetic mutation (e.g., Cre recombinase enzymes and guide-RNA).
• Connect to Experiment – Discuss the importance of these processes in creating prostate cancer mouse models, demonstrating genetic alteration and cancer progression.

• What is the CRISPR/Cas9 knock-in mouse model and how does it assist in genetic research?
• How does the incorporation of GFPs help in tracing cancer progression?
• What is the role of Cas9 endonucleases in the genetic mutation process?

• Practical Applications – Discuss real-world use cases of these techniques in genetic research and cancer study (e.g., prostate cancer research).
• Industry Impact – Identify research sectors benefiting from mouse models, including genome science, biotechnology, and healthcare (e.g., CRISPR technology).
• Societal Importance – Emphasize the wider benefits of this technology, in aiding our understanding of diseases and therapies (e.g., cancer research).
• Link to Scientific Advancements – Discuss the significant breakthroughs these models have led to in scientific research.