電気穿孔法によるマウス膀胱尿路上皮にプラスミド DNA 配信法

The overall goal of this method is to deliver DNA plasmids into the bladder urothelium of live mice in vivo for gene overexpression or genome editing. This method can help answer key questions in the bladder cancer field, such as how do different genetic mutations promote bladder cancer progression? The main advantage of this technique is that it provides a fast and convenient way for delivering genes into bladder without the safety concerns and technical barriers associated with viral vectors.

I first had the idea for this method of trying to use any engineered associated viruses for DNA delivery, which didn't work well the bladder. The implication of this technique can also extend towards gene therapy because electroporation can achieve efficient delivery of DNA plasmids into the urothelial cells. Demonstrating the procedure will be Ofir Stefanson and Yueli Liu from my laboratory.

To begin this procedure, autoclave all surgical instruments and sterilize the workspace with 70%ethanol. Spray surgical instruments and the gloves with 70%ethanol frequently when performing the subsequent procedures to maintain sterile conditions. To begin, anesthetize the mouse and shave the surgical area.

To deplete the urine, apply lubricant to the catheter and ensure that its outer surface is fully covered. Next, insert the catheter into the urethra opening of the mouse and slowly push it until it reaches the bladder. Gently press the abdomen to help urine depletion.

Subsequently, discard the urine with a pipette into a waste beaker, then pipette 80 microliters of PBS into the outer end of the catheter with the other end still in the bladder. Next, carefully attach a one millimeter syringe to the catheter. Gently push the syringe to inject PBS into the bladder.

Leave some PBS in the catheter to avoid creating air bubbles in the bladder. Afterward, remove the syringe and wait for PBS to drain out of the bladder. Gently press the abdomen to evacuate PBS, then discard the PBS in the catheter with a pipette into the waste beaker.

Repeat PBS washing for two more times. For each bladder to transfect, prepare at least 20 microliters of DNA plasmid at one microgram per microliter. Next, add one microliter of trypan blue to the 20 microliters of plasmid solution and a 1.5 milliliter microcentrifuge tube and pipette to mix well.

To deliver the plasmid, apply 70%ethanol to the abdomen. Make a vertical incision of one centimeter to open the abdominal skin above the bladder. Next, cut the muscle layer to expose the bladder.

Pipette at least 20 microliters of plasmid plus trypan blue solution into the outer end of the catheter and detach it to the syringe. Then, inject the plasmid solution into the bladder. If the bladder turns blue, the injection is successful.

Leave some liquid in the catheter to avoid creating air bubbles in the bladder. Afterward, grab the external urethral oraifice and remove the catheter and syringe. Use a string to tighten the external urethral orifice.

Make two loops with the string, and then tie with two knots. This is a crucial step that requires two people to cooperate. Make sure the knots are tight so the plasmids will not leak out.

Now, turn on the electroporation generator. Pinch the bladder with two electrodes and push the foot pedal to perform electroporation. To keep the bladder propped one can pinch the back of the abdominal incision with tweezers.

Avoid any contact between tweezers and the electrodes as this may generate a spark. When it is done, suture the abdominal and skin opening with sterile, surgical suture. Remove the string and apply clips.

Apply iodine onto the wound, and then remove the animal from the isoflurane system. Administer buprenorphine analgesic subcutaneously to alleviate post surgical pain. Keep the animal on the heating pad and return it to the home cage after full recovery.

Check the animal at least once daily for normal mobility, drinking and feeding behaviors, and no signs of infection until the incision is healed. Then, a week later, remove the clips. Administer subsequent injections of buprenorphine analgesic if the animal displays pain symptoms such as reduced grooming, increased aggressiveness, and vocalization.

To demonstrate successful gene delivery into the bladder urothelium, 20 microliters of pCAG-GFP plasmid and trypan blue solution was injected through the urethra and five electric pulses were administered. Shown here is the successful electroporation that turned the bladder green after 48 hours, whereas a negative control bladder that did not undergo electroporation showed no GFP signal. Immunofluorescent staining of sectioned bladder tissues was performed with CK5, CK18, and GFP antibodies.

GFP signal was observed to be present in umbrella, intermediate, and basal cells, but not at the muscle layer indicating good specificity of plasmid delivery into the urothelium. Once mastered, this technique can be performed in 15 minutes if performed properly. After its development, this technique paves the way for researchers in the field of bladder cancer to build new autochthonous mouse models to study disease progression mechanisms.