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

To begin, prep an anesthetized female mouse in the supine position. Ensure its bladder is completely empty. Through the urethral opening, insert a lubricated catheter. Lubrication aids in smooth urethral passage for entry into the bladder.

Following catheterization, make an abdominal incision and dissect the inner musculature to expose the bladder. Next, pipette a suspension of DNA plasmids mixed with a suitable dye into the outer end of the catheter. These DNA plasmids code for a fluorescent reporter gene.

Attach a syringe to the catheter and push it to deliver the plasmid solution into the bladder. The injected dye molecules color the bladder, confirming the successful administration of plasmid solution. Remove the catheter assembly and tighten the external urethral orifice to prevent the plasmid solution out-flow.

Now, pinch the bladder using two electrodes connected to an electroporation generator. Apply electrical pulses of appropriate voltage for a desired period to the bladder. The exposure of the bladder urothelium, the innermost lining of the bladder, to the applied electric fields results in temporary pores in the urothelial cell membrane.

The increased membrane permeability allows the efficient delivery of the injected DNA plasmids into the cells. Following electroporation, suture the surgical incision. Allow the mouse to recover. The urothelial cells transfected with the DNA plasmids express fluorescent reporter proteins.

For each bladder to transfect, prepare at least 20 microliters of DNA plasmid at 1 microgram per microliter. Next, add 1 microliter of Trypan Blue to the 20 microliters of plasmid solution in 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 1 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 attach 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 orifice 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 might 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.