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

Overview

This video demonstrates the delivery of DNA plasmid into the urothelium of the mouse bladder using a combination of urethral catheterization and electroporation techniques. The described procedure allows the generation of mouse bladder cancer models.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
BD 1mL TB Syringe	Fisher	148232E
Buprenorphine	Sigma	B9275-50MG
Dumont Tweezers	Roboz Surgical Instr	RS-5005	Treat with 70% ethanol before surgical use
ECM830 SQ Wave Electroporator	Harvard Apparatus	450052
Exel International Disposable Safelet I.V. Catheters	Fisher Scientific	14-841-21	24G, 2.11 cm length, 0.045 cm outer diameter
Extra Fine Micro Dissecting Scissors	Roboz Surgical Instr	RS-5135	Treat with 70% ethanol before surgical use
Isoflurane	Vet one	501017
K&H Heated Resting Mat for Small Animals, 9 by 12 Inches	Amazon		Cover the heat mat with paper towels
Ophthalmic ointment	Fisher	NC0849514
PBS, pH 7.4	Life Technologies	10010049
Pipet tips 200 µL	USA Scientific	1111-0206
Pipet Tips, Universal Fit; 0.1 to 10 µL	Fisher	02707438(CS)
PIPETMAN NEO P2	Gilson	F144561
PIPETMAN NEO P200N	Gilson	F144565
plasmid CAG-GFP	Addgene	16664
Platinum tweezertrode 5 mm	Harvard Apparatus	450489	5 mm diameter
Scissors	Roboz Surgical Instr	RS-5880	Treat with 70% ethanol before surgical use
String cord	Amazon		Mandala Crafts 150D 210D 0.8mm 1mm leather sewing stitching flat waxed thread string cord
Tape, Scotch	Fisher	19047257
Therio-gel Veterinary Lubricant	PBS animal health	353-736
Trypan Blue Stain	Life Technologies	15250061
V-1 Table top system anesthesia	VetEquip	901806
Vicryl violet suture 18" FS-2 cutting 5-0	Fisher	NC0578475
Walgreens Povidone Iodine 10%	Walgreens	953982
Wound clip	Fisher	18045
Wound clip applier	Fisher	1804