Delivery of DNA Plasmids into Urothelium of Mouse Bladder: An Electroporation-based Procedure to Efficiently Deliver DNA Plasmids into Urothelial Cells of Murine Bladder to Generate Genetically Engineered Models

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Plasmid and Tools Preparation

1. For each bladder to transfect, prepare at least 20 µL of DNA plasmid (1 µg/µL).
2. Add 1 µL of Trypan Blue to the 20 µL of plasmid solution in a 1.5 mL microcentrifuge tube. Pipette to mix well.
3. Autoclave all surgical instruments and sterilize the workspace with 70% ethanol. Spray surgical instruments and gloves with 70% ethanol frequently or use a glass bead sterilizer when performing the following steps to maintain sterile conditions.

2. Plasmid Delivery

1. Apply 70% ethanol to the anesthetized mouse abdomen and use a scalpel to make a vertical incision of 1 cm to open the abdominal skin above the bladder.
2. Pipette at least 20 µL of plasmid plus Trypan Blue solution into the outer end of the catheter and attach the syringe.
3. 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.
4. Grab the external urethral orifice using tweezers and then 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.
   NOTE: Tightening of the urethral is important for preventing backflow of plasmid liquid.
5. Turn on the electroporation generator with the following parameters: 33V, 50 ms working time, and 950 ms interval time.
6. Hold the bladder with tweezers and pinch the bladder with two electrodes. Push the foot pedal to perform the electroporation.
   NOTE: The electric pulses are set to occur 5 times with short intervals after each pedal push. The mouse may twitch in this process. Pushing the foot pedal more than once can increase the delivery efficiency, but too many electric pulses can damage the tissue integrity of the urothelium.
   1. Avoid any contact between the tweezers and the electrodes, as this will generate a spark.

3. Recovery

1. Suture the abdominal and skin opening with sterile surgical sutures and clips. Apply iodine onto the wound.
2. Remove the animal from the isoflurane system. Administer buprenorphine analgesic (0.1 mg/kg) subcutaneously to alleviate post-surgical pain.
3. 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, and then remove the clips. Administer subsequent injections of buprenorphine analgesic if the animal displays pain symptoms such as reduced grooming, increased aggressiveness, and vocalization.