This protocol describes methods that allow investigators to express CDNAs or SIRNAs in the native bladder urothelium. The main advantages of this technique are its relative simplicity, the ability to express cDNAs, siRNAs in the urothelium with high efficiency, and within a relatively short period of time. The hardest part of this technique is the catheterization.
However, the overall technique is relatively easy to master once learned. Demonstrating the procedure will be Giovanni Ruiz, a Research Specialist IV in my laboratory. To begin place the anesthetized mouse on a heating pad next to the nose cones.
Maintain the animal under anesthesia for the duration of the protocol. Confirm the mouse is completely anesthetized with a toe pinch. To prevent introducing air into the bladder, fill the plastic catheter portion of an IV catheter and associated hub with sterile PBS using a sterile transfer pipette or syringe.
With the animal in the supine position swab the external urethral meatus with 70%alcohol. Then gently grab the tissue forming the external urethral meatus and extend it vertically away from the animal using fine forceps. Insert the sterile catheter into the external meatus.
Using the other hand carefully insert the catheter vertically about three to four millimeters into the urethral meatus. Then lower the catheter inserted into the external meatus toward the animal's tail, which eases its entry into the portion of the urethra that passes below the pubic bone and ultimately into the bladder. Allow the urine in the bladder to leak out.
Remove any residual urine by performing Crede's maneuver by massaging and gently pressing down on the bladder bump in the lower abdominal area. To make the urothelium receptive to transduction, wash the mouse or rat bladder by attaching a sterile one milliliter syringe filled with PBS to the catheter hub. Inject 100 microliters of sterile PBS into the mouse bladder.
After detaching the syringe from the catheter fitting, allow the PBS to drain. Instill 100 microliters of 0.1%N-dodecyl-beta-D-maltoside dissolved in PBS and 0.2 micrometer filter sterilized into the mouse bladder using a sterile one-milliliter syringe. Retain the N-dodecyl-beta-D-maltoside in the bladder for 10 minutes by leaving the syringe in place.
Remove the N-dodecyl-beta-D-maltoside from the bladder by detaching the syringe and allowing it to drain out. To introduce the virus into the bladder attach a sterile one-milliliter syringe to the catheter hub and instill 5, 000, 000 to 100, 000, 000 IVP of adenovirus diluted in 100 microliters of sterile PBS for mice, or 450 microliters for rats into the bladder. After 30 minutes, detach the syringe and allow the virus solution to evacuate the bladder onto a disposable pad.
Blot any residual virus solution with an absorbent wipe while discarding the pad and wipe in biohazardous waste. In order to allow the animal to recover, cease the flow of isoflurane. Allow the animal to recover and be fully mobile before returning it to its cage, particularly if the animals are group-housed.
Analyze the effects of transgene expression 12 to 72 hours post-treatment using methods such as mRNA in situ hybridization, Western blot, or immunofluorescence. Western blot analysis of urothelial lysates revealed V5-tagged human growth hormone expression in the urothelium of transduced bladders, but not in untransduced ones. The expression was also confirmed by immunofluorescence using antibodies that recognized human growth hormone, or the V5 epitope tag.
Western blot analysis confirmed the expression of Claudin-2 in rats transduced with adenovirus, but not in those transduced with a controlled GFP-encoding virus. Immunofluorescence analysis further confirmed exogenous Claudin-2 expression in urothelial umbrella cells transduced with virus-encoding Claudin-2 cDNA. Detection of exogenous Claudin-2 in Tight Junction Protein 1 by immunofluorescence and confocal microscopy of whole mounted or cross-sectioned urothelium revealed the presence of Tight Junction and intracellular accumulations of Claudin-2 in the cell cytoplasm.
The image field shows that counting the number of transduced umbrella cells reveals an efficiency that approaches 95%and in the entirety of the bladder wall only the urothelium is transduced, and no other tissue is targeted by the instilled adenovirus. The catheterization is critical for the protocol to work as intended. This technique allows investigators to study normal urothelial biology, as well as to understand conditions where protein overexpression, or underexpression, leads to disease.
