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.
