This work was supported by a pilot project award through P30DK079307 (to M.G.D.), NIH grant R01DK119183 (to G.A. and M.D.C.), NIH grant R01DK129473 (to G.A.), an American Urology Association Career Development award and a Winters Foundation grant (to N.M.), by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307), and by S10OD028596 (to G.A.), which funded the purchase of the confocal system used to capture some of the images presented in this manuscript.

Experiments involving the generation of adenoviruses, which requires BSL2 certification, were performed under approval from the University of Pittsburgh Environmental Health and Safety offices and the Institutional Biosafety Committee. All animal experiments performed, including adenoviral transduction (which requires ABSL2 certification), were done in accordance with relevant guidelines/regulations of the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Animal Welfare Act, and under the approval of the University of Pittsburgh Institutional Animal Care and Use Committee. Gloves, eye protection, and appropriate garb are worn for all procedures involving recombinant viruses. Any liquid or solid waste should be disposed of as described below. The bedding of the animals post transduction, and any resulting animal carcasses, should be treated as biohazardous materials and disposed of according to institutional policies.
1. Preparation of high-titer adenovirus stocks
NOTE: Effective transduction of rodent bladders depends on the use of purified and concentrated viral stocks, typically 1 x 107 to 1 x 108 infectious viral particles (IVP) per &#181;L. This portion of the protocol is focused on generating high-titer adenovirus stocks from existing virus preparations. All steps should be performed in a cell culture hood using sterile reagents and tools. While the available strains of adenovirus used today are replication defective, most institutions require approval to use adenoviruses and recombinant DNA. This often includes designation of a cell culture room as a BSL2 approved facility to produce and amplify adenoviruses. Some general considerations include use of masks, eye protection, gloves, and appropriate garb at all stages of virus production and purification. When performing centrifugation, safety caps are recommended if the centrifuge tubes lack tight-fitting caps. All non-disposable materials, including potentially contaminated centrifuge safety caps, bottles, and rotors are treated with an antiviral solution (see Table of Materials), and then rinsed with water or 70% ethanol. Liquid wastes are treated by adding bleach to a final concentration of 10% (v/v). Disposal of these liquid wastes will depend on institutional policies. Solid wastes are typically disposed of in biohazardous waste.
<ol>
	<li>Culture HEK293T cells
	<ol>
		<li>Thaw a frozen vial of HEK293T cells in a water bath at 37 &#176;C and use a 5 mL pipette to transfer the cells to a 15 cm diameter cell culture dish. Using a 25 mL pipette, slowly add 20 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum and penicillin/streptomycin antibiotic (DMEM-FBS-PS) to the dish. Incubate the cells in a 37 &#176;C cell-culture incubator gassed with 5% (v/v) CO2 until they reach 80%-90% confluence (~2 x 107 cells).</li>
		<li>Using a glass pipette attached to a vacuum source, aspirate the medium, and then rinse the cells by transferring 20 mL of sterile PBS (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, and 8.9 mM Na2HPO4) to the dish using a 25 mL pipette. Aspirate the spent PBS, and then use a 5 mL pipette to transfer 3 mL of warm (37 &#176;C) proteinase solution (see Table of Materials) to the dish, incubating the dish in the cell-culture incubator until the cells detach (~3-4 min). 
		NOTE: Effective proteolysis can be best assessed by slowly tilting the dish back and forth looking for release of cells from all portions of the dish into the moving fluid. HEK293T cells are sensitive to extended proteinase treatment and will die if left more than a few minutes in proteinase solution.</li>
		<li>Use a 10 mL pipette to transfer 7 mL of DMEM-FBS-PS to the dish of detached cells, and then use the same pipette to aspirate the cells and medium. Transfer the suspended cells to a 50 mL conical tube, and then pellet the cells by centrifuging the suspension in a low-speed clinical centrifuge at 200 x g for 5 min. Aspirate the supernatant using a glass pipette attached to a vacuum source. Use a 25 mL pipette to resuspend the cell pellet in 15 mL of DMEM-FBS-PS.</li>
		<li>Add 1 mL of the cell suspension to each of fifteen 15 cm dishes containing 19 mL of DMEM-FBS-PS.</li>
		<li>Grow the cells in a tissue culture incubator until they reach 85%-90% confluence (~3-4 days).</li>
	</ol>
	</li>
	<li>Prepare a diluted virus solution
	<ol>
		<li>Add ~1.5 x 109 to 3 x 109 IVP of an existing virus stock (5-10 IVP/cell) to a 50 mL conical tube filled with 45 mL of DMEM lacking FBS or antibiotics. 
		NOTE: It is possible to use a lower concentration of the virus (1-5 IVP/cell); however, it will take longer for virus production to ensue.</li>
	</ol>
	</li>
	<li>Infect the cultured cells with the virus.
	<ol>
		<li>Using a glass pipette attached to a vacuum source, aspirate the medium from the almost confluent cells in step 1.1.5.</li>
		<li>Use a 25 mL pipette to transfer 3.0 mL of diluted virus solution (prepared in step 1.2.1) to each cell-culture dish. Then, use a 10 mL pipette to add 7.0 mL of DMEM medium (lacking FBS or antibiotics) to each dish. Incubate for 60 min in a cell-culture incubator, and then add 10 mL of DMEM containing 20% (v/v) FBS and 2x PS to each dish. 
		NOTE: Using one of the 15 dishes as a control plate (in which virus is not added) makes it easier to identify virus-induced cell rounding and death in infected cells in the next step.</li>
		<li>Incubate the cells in the cell culture incubator for 2-4 days until the majority of them begin to round up and &#62;60% of the cells have detached. 
		NOTE: If using a lower titer of the virus, it may take up to a week for cell death to occur. If cell death does not occur within a week, it is likely the process will need to be repeated using a higher titer of the virus.</li>
	</ol>
	</li>
	<li>Recover virus from cell lysates
	<ol>
		<li>Use a cell scraper to scrape the bottom of each dish, releasing attached cells into the medium.</li>
		<li>Using a 25 mL pipette, collect and pool the medium, cells, and cell debris from each cell culture dish into a 50 mL conical cell culture tube. 
		NOTE: To save resources, the medium from two dishes can be combined into one 50 mL tube.</li>
		<li>Pellet the cellular material by centrifugation using a low-speed table-top centrifuge: 5 min at room temperature at 3,000 x g. Use a glass pipette attached to a vacuum device to aspirate the supernatant.</li>
		<li>Use a 10 mL pipette, combined with trituration, to consolidate all the resulting pelleted material in a total of 7 mL of sterile-filtered 100 mM Tris-HCl pH 7.4 containing 10 mM EDTA (Tris-EDTA solution). Transfer the pooled material to a sterile 15 mL cell culture conical tube and place on ice. 
		NOTE: At this point, the virus prep can be frozen at -80 &#176;C indefinitely.</li>
	</ol>
	</li>
	<li>Prepare cell lysates
	<ol>
		<li>Perform three freeze-thaw cycles to disrupt the remaining cells, and thus further liberate the formed virus particles. Freeze the pooled material in step 1.4.4 by submerging the tube in liquid nitrogen (~30-60 s). Rapidly thaw the sample by placing the tube in a 37 &#176;C incubator. Vortex the sample for 15 s, and then repeat the rapid freezing and thawing procedure an additional 2x. 
		NOTE: The virus solution can be rapidly thawed in a 37 &#176;C in a water bath, but caution is warranted as the temperature swings can cause the tube to crack, releasing virus solution into the water bath. To prevent this from occurring, place the tube containing the viral supernatant into a larger tube, which is then set in the water bath.</li>
		<li>Use a 10 mL pipette to transfer the thrice freeze-thawed cellular material to a superspeed centrifuge tube. Centrifuge the material in the tube for 30 min at 4 &#176;C super-speed centrifuge at ~18,500 x g.</li>
		<li>Recover the ~7 mL of virus-rich supernatant with a 10 mL pipette and transfer it to a 15 mL conical tube. Retain the sample on ice until the next step. 
		NOTE: At this point, consider keeping an aliquot of the unpurified viral supernatant as a preamble to start a new virus preparation (or as a backup). Store this aliquot at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Isolate and purify the virus using density gradient centrifugation.
	<ol>
		<li>Prepare a discontinuous gradient of CsCl in a 12 mL PET thin-wall, clear ultracentrifuge tube (see Table of Materials) or equivalent. Use a 3 mL syringe outfitted with an 18 G needle to carefully introduce 2.5 mL of 1.4 g/mL CsCl solution into the bottom of the tube, and then use a new syringe/needle to layer it with 2.5 mL of 1.25 g/mL CsCl solution. 
		NOTE: Dropping solutions directly on top of a preexisting layer will cause significant and unwanted mixing. Instead, place the beveled portion of the needle against the edge of the tube, and then very slowly press the syringe plunger, raising the position of the needle as the solution fills the tube.</li>
		<li>Load the ~7 mL of viral supernatant on top of the gradient in a similar fashion using a 10 mL syringe. If there is more than 2-3 mm of space between the viral supernatant and the top of the tube, add additional Tris-EDTA solution to fill the tube until only 2-3 mm of space remains.</li>
		<li>Make a similarly prepared balance tube, containing layers of CsCl, but substituting Tris-EDTA solution for the viral supernatant. 
		NOTE: The two tubes must have identical weights (and similar densities) to prevent a potentially dangerous unbalanced load situation in the ultracentrifuge.</li>
	</ol>
	</li>
	<li>Isolate the virus using rate-zonal ultracentrifugation.
	<ol>
		<li>Load the gradients formed in steps 1.6.2-1.6.3 into the buckets of an SW41 rotor or equivalent. Screw in bucket caps, place the rotor into an ultracentrifuge (precooled to 4 &#176;C), and centrifuge for 1 h at ~150,000 x g.</li>
		<li>During centrifugation, equilibrate the column described in step 1.9.1 below.</li>
	</ol>
	</li>
	<li>Recover isolated virus particles
	<ol>
		<li>At the end of the centrifugation step, carefully detach the buckets from the rotor, and in a cell-culture hood, remove the bucket caps, and then remove the tubes and place them in a rack.</li>
		<li>Collect the banded material, rich in virus particles, that floats at the interface between the 1.25 g/mL and 1.4 g/mL CsCl solution. Holding the tube containing the gradient over the bottom half of a cell culture dish (which will catch any spilled materials), use a 1 inch 18 G needle attached to a 3 mL syringe to carefully puncture the tube, just below the banded virus. Slowly aspirate the virus, which is typically recovered in ~1 mL.</li>
		<li>Remove the needle from the tube, which will result in the remaining material in the gradient to flow out of the tube into the bottom half of the cell-culture dish (any liquids should be treated as hazardous waste).</li>
		<li>Transfer the virus solution in the syringe into a sterile microcentrifuge tube on ice. 
		NOTE: When recovering the virus in this step, position the needle so that the lumen of the needle is facing upward with&#160;the needle opening a few mm below the virus-rich band. Avoid contamination by the thin band of improperly assembled virus that is sometimes observed 2-3 mm above the banded virus (see thin black arrow in Figure 1A).</li>
	</ol>
	</li>
	<li>Remove CsCl from the sample by gel filtration.
	<ol>
		<li>Equilibrate a PD-10 column (pre-packed with Sephadex G-25M), clamped to a support stand, with 50 mL of 0.2 &#181;m sterile-filtered PBS containing 10% (v/v) glycerol. 
		NOTE: The initial equilibration step takes 2-3 h to complete and is necessary to ensure complete washout of preservatives that are used by the manufacturer to stabilize the column.</li>
		<li>Allow the wash solution to recede below the frit (a protective disc of white material at the top of the column medium), and then carefully transfer the purified virus solution collected in step 1.8 to the top of the column. Allow the virus-rich solution to recede below the frit, and then begin filling the column with PBS-glycerol. 
		NOTE: One function of the frit is to prevent the column from running dry. As a result, short delays are tolerated before more eluant is added to the column.</li>
		<li>Collect the eluate in 12 sterile microcentrifuge tubes, 0.5 mL per fraction.</li>
	</ol>
	</li>
	<li>Determine the viral yield.
	<ol>
		<li>Determine the peak virus fractions using spectrophotometry. Prepare a 1:100 dilution of each fraction in PBS and measure the OD260 in a spectrophotometer, using a 1:100 dilution of buffer alone as a blank. The virus particles should elute in the void volume, starting around fraction 6. Pool the fractions containing the highest OD260 readings.</li>
		<li>Make a 1:100 dilution of the pooled viral fractions and remeasure the OD260. Calculate the final concentration of virus particles and the number of IVP in the pooled fractions using the following formula: Virus particles per mL = OD260 &#215; 100 (this corrects for the dilution factor) &#215; 1012. A general estimate is that 1% of the virus particles are IVP, as such: IVP/mL = OD260 &#215; 100 &#215; 1010 or IVP/&#181;L = OD260 &#215; 100 &#215; 107.</li>
	</ol>
	</li>
	<li>Aliquot the pooled virus fractions (containing 5 x 107 to 1 x 108 IVP) into sterile microcentrifuge tubes or cryovials. Store the samples at -80 &#176;C.</li>
</ol>
2. Transduction of rodent bladder
NOTE: If new to this technique, it is recommended that the number of animals transduced at one time be limited to 2-4. This can be accomplished by staggering the start times for each animal, particularly during the detergent treatment in step 2.2, and then the virus incubation in step 2.3. Experienced investigators can transduce up to six animals at a time.
<ol>
	<li>Catheterize the bladder
	<ol>
		<li>Anesthetize female C57Bl/6J mice (typically 8-10 weeks old, ~20-25 g) or female Sprague Dawley rats (typically 2-3 months old, ~250 g) using a vaporizer with an attached nose cone. Calibrate the vaporizer to produce 3.0% (v/v) isoflurane, 97% (v/v) O2 for mice or 4.0% (v/v) isoflurane, 96% (v/v) O2 for rats. Confirm that the animals are anesthetized by ensuring they are unresponsive to toe pinch (typically after 1-2 min).</li>
		<li>Maintain the animal&#39;s body temperature by placing the animals on a heated pad. Monitor the animal throughout the transduction protocol to ensure the animals are anesthetized and do not experience any pain during this procedure.</li>
		<li>Reduce isoflurane to 1.5% (v/v) for mice or 2.0% (v/v) for rats and maintain the animals under anesthesia for the duration of the protocol.</li>
		<li>To prevent introducing air into the bladder, fill the plastic catheter portion of an IV catheter (see Table of Materials) and associated hub with sterile PBS using a transfer pipette.</li>
		<li>With the animal in the supine position, swab the external meatus with 70% alcohol, and insert the sterile catheter into the external meatus, then the urethra, and then into the bladder.
		<ol>
			<li>To perform this task, use fine forceps to gently grab the tissue forming the external meatus and extend it vertically, away from the animal. Using the other hand, carefully insert the catheter vertically about 3-4 mm into the urethral meatus (a mound of flesh just above the opening of the vagina). Then, because of a bend in the urethra, lower the catheter, inserted into the external meatus, toward the tail of the animal, which eases its entry into the portion of the urethra that passes below the pubic bone, and ultimately into the bladder 
			.NOTE: Especially in the case of the mouse, the catheter may be too long, and no more that 1.0-1.1 cm of it should be inserted into the animal. Otherwise, damage to the bladder mucosa will ensue. To prevent this, mark the catheter ~1 cm below its tip, and then do not insert the catheter beyond this marking.</li>
		</ol>
		</li>
		<li>Allow the urine in the bladder to leak out. Remove any residual urine by performing Cred&#233;&#39;s maneuver: massage and gently press down on the bladder bump in the lower abdominal area.</li>
	</ol>
	</li>
	<li>Make the urothelium receptive to transduction by treating it with a detergent solution.
	<ol>
		<li>Wash the mouse or rat bladder by attaching a sterile 1 mL syringe filled with PBS to the catheter hub. Inject 100 &#181;L of sterile PBS into the mouse bladder (or 450 &#181;L in the case of the rat bladder). Detach the syringe from the catheter fitting and allow the PBS drain. If necessary, perform Crede&#39;s maneuver to remove excess bladder fluid.</li>
		<li>Instill 100 &#956;L of 0.1% (w/v) N-dodecyl-&#946;-D-maltoside (DDM) (dissolved in PBS and 0.2 &#181;m filter-sterilized) into the mouse bladder using a sterile 1 mL syringe. Retain the DDM in the bladder for 10 min by leaving the syringe in place. In rats, the volume of DDM is increased to 450 &#181;L.</li>
		<li>Remove the DDM from the bladder by detaching the syringe and allowing it to drain out. Perform Crede&#39;s maneuver if necessary.</li>
	</ol>
	</li>
	<li>Introduce the virus into the bladder.
	<ol>
		<li>Attach a sterile 1 mL syringe to the catheter hub and instill 0.5 x 107 to 1 x 108 IVP of adenovirus (prepared in step 1), diluted in 100 &#181;L of sterile PBS for mice or 450 &#181;L for rats, into the bladder. Leave the syringe attached to the catheter to prevent the virus solution from escaping.</li>
		<li>After 30 min, 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, and discard the pad and wipe in biohazardous waste. 
		NOTE: Glycerol is cytotoxic. As such, the maximal volume of virus solution instilled in mice is limited to 5 &#181;L (which when diluted into 100 &#181;L of PBS would result in a final glycerol concentration of ~0.5% [v/v]). Additionally, it is possible to enhance transduction efficiency by increasing the number of instilled IVP and by extending the virus incubation to 45 min.</li>
		<li>Optional step: The bladder can be rinsed with PBS as described in step 2.2.1 above; however, this is not required.</li>
	</ol>
	</li>
	<li>Allow the animal to recover.
	<ol>
		<li>Cease the flow of isoflurane and allow the animal to recover and be fully mobile before returning it to its cage, particularly if the animals are group housed. 
		NOTE: As virus transduction per se does not cause observable lower urinary tract symptoms or pain, there is usually no need for post-surgical treatment. However, if the encoded transgene is toxic, post-procedure analgesia or antibiotics may be necessary as required by the institution.</li>
	</ol>
	</li>
	<li>Analyze the effects of transgene expression 12-72 h post treatment using methods such as mRNA in situ hybridization, western blot, or immunofluorescence9,10,23 (see the representative results).</li>
</ol>

<ol>
	<li>Dalghi, M. G., Montalbetti, N., Carattino, M. D., Apodaca, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Urothelium:+Life+in+a+liquid+environment.">The Urothelium: Life in a liquid environment.</a> Physiological Reviews. 100 (4), 1621-1705 (2020).</li><li>Truschel, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-regulated+exocytosis/endocytosis+in+bladder+umbrella+cells.">Stretch-regulated exocytosis/endocytosis in bladder umbrella cells.</a> Molecular Biology of the Cell. 13 (3), 830-846 (2002).</li><li>Lewis, S. A., de Moura, J. L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+cytoplasmic+vesicles+into+apical+membrane+of+mammalian+urinary+bladder+epthelium.">Incorporation of cytoplasmic vesicles into apical membrane of mammalian urinary bladder epthelium.</a> Nature (London). 297 (5868), 685-688 (1982).</li><li>Eaton, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+and+contraction+of+the+umbrella+cell+apical+junctional+ring+in+response+to+bladder+filling+and+voiding.">Expansion and contraction of the umbrella cell apical junctional ring in response to bladder filling and voiding.</a> Molecular Biology of the Cell. 30 (16), 2037-2052 (2019).</li><li>Yu, W., Khandelwal, P., Apodaca, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+apical+and+basolateral+membrane+requirements+for+stretch-induced+membrane+traffic+at+the+apical+surface+of+bladder+umbrella+cells.">Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells.</a> Molecular Biology of the Cell. 20 (1), 282-295 (2009).</li><li>Birder, L., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+signaling.">Urothelial signaling.</a> Physiological Reviews. 93 (2), 653-680 (2013).</li><li>Birder, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+bladder,+cystitis+and+nerve/urothelial+interactions.">Urinary bladder, cystitis and nerve/urothelial interactions.</a> Autonomic Neuroscience. 182, 89-94 (2014).</li><li>Apodaca, G., Balestreire, E., Birder, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uroepithelial-associated+sensory+web.">The uroepithelial-associated sensory web.</a> Kidney International. 72 (9), 1057-1064 (2007).</li><li>Dalghi, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+for+PIEZO1+and+PIEZO2+in+urothelial+mechanotransduction+and+lower+urinary+tract+interoception.">Functional roles for PIEZO1 and PIEZO2 in urothelial mechanotransduction and lower urinary tract interoception.</a> Journal of Clinical Investigation Insight. 6 (19), 152984 (2021).</li><li>Montalbetti, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+urothelial+paracellular+transport+promotes+cystitis.">Increased urothelial paracellular transport promotes cystitis.</a> American Journal of Physiology: Renal Physiology. 309 (12), 1070-1081 (2015).</li><li>Keay, S. K., Birder, L. A., Chai, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+bladder+urothelial+pathophysiology+in+functional+bladder+disorders.">Evidence for bladder urothelial pathophysiology in functional bladder disorders.</a> BioMed Research International. 2014, 865463 (2014).</li><li>Friedel, R. H., Wurst, W., Wefers, B., Kuhn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+conditional+knockout+mice.">Generating conditional knockout mice.</a> Methods in Molecular Biology. 693, 205-231 (2011).</li><li>Kashyap, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+nerve+growth+factor+expression+in+the+bladder+by+antisense+oligonucleotides+as+new+treatment+for+overactive+bladder.">Down-regulation of nerve growth factor expression in the bladder by antisense oligonucleotides as new treatment for overactive bladder.</a> Journal of Urology. 190 (2), 757-764 (2013).</li><li>Bolenz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+uptake+and+ex+vivo+urothelial+penetration+by+oligodeoxynucleotides+for+optimizing+treatment+of+transitional+cell+carcinoma.">Cellular uptake and ex vivo urothelial penetration by oligodeoxynucleotides for optimizing treatment of transitional cell carcinoma.</a> Analytical and Quantitative Cytology and Histology. 30 (5), 265-273 (2008).</li><li>Tyagi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravesical+antisense+therapy+for+cystitis+using+TAT-peptide+nucleic+acid+conjugates.">Intravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugates.</a> Molecular Pharmaceutics. 3 (4), 398-406 (2006).</li><li>Sadoff, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interim+results+of+a+phase+1-2a+trial+of+Ad26.COV2.S+Covid-19+vaccine.">Interim results of a phase 1-2a trial of Ad26.COV2.S Covid-19 vaccine.</a> New England Journal of Medicine. 384 (19), 1824-1835 (2021).</li><li>Lee, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+delivery:+Potential+applications+for+gene+and+cell-Based+therapies+in+the+new+era+of+personalized+medicine.">Adenovirus-mediated gene delivery: Potential applications for gene and cell-Based therapies in the new era of personalized medicine.</a> Genes &amp; Diseases. 4 (2), 43-63 (2017).</li><li>Ramesh, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pretreatment+agents+to+enhance+adenovirus+infection+of+bladder+epithelium.">Identification of pretreatment agents to enhance adenovirus infection of bladder epithelium.</a> Molecular Therapy. 10 (4), 697-705 (2004).</li><li>Khandelwal, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab11a-dependent+exocytosis+of+discoidal/fusiform+vesicles+in+bladder+umbrella+cells.">Rab11a-dependent exocytosis of discoidal/fusiform vesicles in bladder umbrella cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15773-15778 (2008).</li><li>Khandelwal, P., Ruiz, W. G., Apodaca, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+endocytosis+in+bladder+umbrella+cells+occurs+through+an+integrin-regulated+and+RhoA-+and+dynamin-dependent+pathway.">Compensatory endocytosis in bladder umbrella cells occurs through an integrin-regulated and RhoA- and dynamin-dependent pathway.</a> The European Molecular Biology Organization journal. 29 (12), 1961-1975 (2010).</li><li>Khandelwal, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rab11a-Rab8a-Myo5B+network+promotes+stretch-regulated+exocytosis+in+bladder+umbrella+cells.">A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells.</a> Molecular Biology of the Cell. 24 (7), 1007-1019 (2013).</li><li>Prakasam, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A1+adenosine+receptor-stimulated+exocytosis+in+bladder+umbrella+cells+requires+phosphorylation+of+ADAM17+Ser811+and+EGF+receptor+transactivation.">A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser811 and EGF receptor transactivation.</a> Molecular Biology of the Cell. 25 (24), 3798-3812 (2014).</li><li>Gallo, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAB27B+requirement+for+stretch-induced+exocytosis+in+bladder+umbrella+cells.">RAB27B requirement for stretch-induced exocytosis in bladder umbrella cells.</a> American Journal of Physiology Cell Physiology. 314 (3), 349-365 (2018).</li><li>Amasheh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claudin-2+expression+induces+cation-selective+channels+in+tight+junctions+of+epithelial+cells.">Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells.</a> Journal of Cell Science. 115, 4969-4976 (2002).</li><li>Yu, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+cation+selectivity+in+claudin-2-based+paracellular+pores:+identification+of+an+electrostatic+interaction+site.">Molecular basis for cation selectivity in claudin-2-based paracellular pores: identification of an electrostatic interaction site.</a> The Journal of General Physiology. 133 (1), 111-127 (2009).</li><li>Kasahara, H., Aoki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+using+adenoviral+RNAi+vector+in+vascular+smooth+muscle+cells+and+cardiomyocytes.">Gene silencing using adenoviral RNAi vector in vascular smooth muscle cells and cardiomyocytes.</a> Methods in Molecular Medicine. 112, 155-172 (2005).</li><li>Bolognesi, B., Lehner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaching+the+limit.">Reaching the limit.</a> eLife. 7, 39804 (2018).</li><li>Atasheva, S., Shayakhmetov, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+responses+to+adenovirus+and+adenovirus+vectors.">Cytokine responses to adenovirus and adenovirus vectors.</a> Viruses. 14 (5), 888 (2022).</li><li>Sanchez Freire, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+may+mediate+the+down-regulation+of+neurokinin-1+receptor+in+chronic+bladder+pain+syndrome.">MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.</a> American Journal of Pathology. 176 (1), 288-303 (2010).</li></ol>

The authors have nothing to disclose.

While Ramesh et al. were focused on developing strategies to use adenoviral transduction in the treatment of bladder cancer18, more recent reports have demonstrated the utility of these techniques in studying normal urothelial biology/physiology and pathophysiology10,18,19,20,21. The important features of this approach include the follo...

The urothelium is the specialized epithelium that lines the renal pelvis, ureters, bladder, and proximal urethra1. It comprises three strata: a layer of highly differentiated and polarized often bi-nucleate umbrella cells, whose apical surfaces are bathed in urine; an intermediate cell layer with a population of bi-nucleate transit-amplifying cells that can give rise to superficial umbrella cells in response to their acute loss; and a single layer of basal cells, a subset of which function as stem cells that can regenerate the entirety of the urothelium in response to chronic injury. Umbrella cells are chiefly responsible for forming the high-resistance urothelial barrier, components of which include an apical membrane (rich in cholesterol and cerebrosides) with low permeability to water and solutes, and a high-resistance apical junctional complex (comprised of tight junctions, adherens junctions, desmosomes, and an associated actomyosin ring)1. Both the apical surface of the umbrella cell and its junctional ring expand during bladder filling and return to their pre-filled state rapidly after voiding1,2,3,4,5. In addition to its role in barrier function, the urothelium is also hypothesized to have sensory and transducer functions that allow it to sense changes in the extracellular milieu (e.g., stretch) and transmit this information via release of mediators (including ATP, adenosine, and acetylcholine) to underlying tissues, including suburothelial afferent nerve processes6,7,8. Recent evidence of this role is found in mice lacking urothelial expression of both Piezo1 and Piezo2, which results in altered voiding function9. Additionally, rats overexpressing the tight-junction pore-forming protein CLDN2 in the umbrella cell layer develop inflammation and pain analogous to that seen in patients with interstitial cystitis10. It is hypothesized that disruption of urothelial sensory/transducer or barrier function may contribute to several bladder disorders6,11.
A better understanding of the biology of the urothelium in normal and disease states depends on the availability of tools that will allow investigators to readily downregulate endogenous gene expression or allow for the expression of transgenes in the native tissue. While one approach to downregulate gene expression is to generate conditional urothelial knockout mice, this approach depends on the availability of mice with floxed alleles, is labor intensive, and can take months to years to complete12. Not surprisingly then, investigators have developed techniques to transfect or transduce the urothelium, which can lead to results on a shorter time scale. Published methods for transfection include the use of cationic lipids13, anti-sense phosphorothioated oligodeoxynucleotides14, or antisense nucleic acids tethered to the HIV TAT protein penetrating 11-mer peptide15. However, the focus of this protocol is on the use of adenoviral-mediated transduction, a well-studied methodology that is efficient at gene delivery to a broad range of cells, has been tested in numerous clinical trials, and most recently was used to deliver the cDNA encoding the COVID-19 capsid protein to recipients of one variant of the COVID-19 vaccine16,17. For a more thorough description of the adenovirus life cycle, adenoviral vectors, and clinical applications of adenoviruses, the reader is directed to reference17.
An important milestone in the use of adenoviruses to transduce the urothelium, was a report by Ramesh et al. that showed short pretreatments with detergents, including N-dodecyl-&#946;-D-maltoside (DDM) dramatically enhanced transduction of the urothelium by an adenovirus encoding &#946;-galactosidase18. Using this proof-of-principle study as a guide, adenoviral-mediated transduction of the urothelium has now been used to express a variety of proteins, including Rab-family GTPases, guanine-nucleotide exchange factors, myosin motor fragments, pore-forming tight junction-associated claudins, and ADAM1710,19,20,21,22. The same approach was adapted to express small interfering RNAs (siRNA), the effects of which were rescued by co-expressing siRNA-resistant variants of the transgene22. The protocol described here includes general methods to generate large amounts of highly concentrated adenovirus, a requirement for these techniques, as well adaptations of the methods of Ramesh et al.18 to express transgenes in the urothelium with high efficiency.

<table><tbody><tr><td>10 mL pipette</td><td>Corning Costar (Millipore Sigma)</td><td>CLS4488</td><td>sterile, serological pipette, individually wrapped</td></tr><tr><td>12 mL ultracentrifuge tube</td><td>ThermoFisher</td><td>06-752</td><td>PET thinwall ultracentrifuge tube</td></tr><tr><td>15 mL conical centrifuge tube</td><td>Falcon (Corning)</td><td>352097</td><td>sterile</td></tr><tr><td>18 G needle</td><td>BD&amp;nbsp;</td><td>305196</td><td>18 G x 1.5 in needle</td></tr><tr><td>20 mL pipette</td><td>Corning Costar (Millipore Sigma)</td><td>CLS4489</td><td>sterile, serological pipette, individually wrapped</td></tr><tr><td>50 mL conical centrifuge tube</td><td>Falcon (Corning)</td><td>352098</td><td>sterile</td></tr><tr><td>5 mL pipette</td><td>Corning Costar (Millipore Sigma)</td><td>CLS4487</td><td>sterile, serological pipette, individually wrapped</td></tr><tr><td>Cavicide</td><td>Henry Schein</td><td>6400012</td><td>Anti-viral solution</td></tr><tr><td>Cell culture dish - 15 cm</td><td>Falcon (Corning)</td><td>353025</td><td>sterile, tissue-culture treated&amp;nbsp; (150 mm x 25 mm dish)</td></tr><tr><td>Cell scraper</td><td>Sarstedt</td><td>893.1832</td><td>handle length 24 cm, blade length 1.7 cm</td></tr><tr><td>CsCl</td><td>Millipore Sigma</td><td>C-4306</td><td>Molecular Biology grade &amp;ge; 98%</td></tr><tr><td>DMEM culture medium (high glucose)</td><td>Gibco (ThermoFisher)</td><td>11965092</td><td>with 4.5 g/L glucose + L-glutamine + phenol red</td></tr><tr><td>EDTA</td><td>Millipore Sigma</td><td>EDS</td><td>Bioiultra grade &amp;ge; 99%</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Hyclone (Cytiva)</td><td>SH30070.03</td><td>defined serum</td></tr><tr><td>Glass pipette</td><td>Fisher Scientific</td><td>13-678-20A</td><td>5.75 in glass pipette, autoclaved</td></tr><tr><td>Glycerol</td><td>Millipore Sigma</td><td>G-5516</td><td>Molecular Biology grade &amp;ge; 99%</td></tr><tr><td>HEK293 cells</td><td>ATCC</td><td>CRL-3216</td><td>HEK293T cells are a variant of HEK293 cells that express the SV40 large T-antigen</td></tr><tr><td>Isoflurane</td><td>Covetrus</td><td>29405</td><td></td></tr><tr><td>IV catheter - mouse</td><td>Smith Medical Jelco</td><td>3063</td><td>24 G x 3/4 in Safety IV catheter&amp;nbsp; radiopaque</td></tr><tr><td>IV catheter - rat</td><td>Smith Medical Jelco</td><td>3060</td><td>22 G x 1 in Safety IV catheter radiopaque</td></tr><tr><td>KCl</td><td>Millipore Sigma</td><td>P-9541</td><td>Molecular Biology grade &amp;ge; 99%</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Millipore Sigma</td><td>P5655</td><td>Cell culture grade&amp;nbsp; &amp;ge; 99%</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Millipore Sigma</td><td>431478</td><td>&amp;nbsp;&amp;ge; 99.99%</td></tr><tr><td>NaCl</td><td>Millipore Sigma</td><td>S3014</td><td>Molecular Biology grade &amp;ge; 99%</td></tr><tr><td>N-dodecyl-&amp;beta;-D-maltoside&amp;nbsp;</td><td>Millipore Sigma</td><td>D4641</td><td>&amp;nbsp;&amp;ge; 98%</td></tr><tr><td>Nose cone for multiple animals</td><td>custom designed</td><td></td><td>commercial options include one from Parkland Scientific (RES3200)</td></tr><tr><td>PD-10 column&amp;nbsp;</td><td>GE Healthcare</td><td>17-085-01</td><td>Prepacked columns filled ith Sephadex G-25M</td></tr><tr><td>Penicillin/streptomycin antibiotic (100x)</td><td>Gibco (ThermoFisher)</td><td>15070063</td><td>100x concentrated solution</td></tr><tr><td>Spectrophotometer</td><td>Eppendorf&amp;nbsp;</td><td>BioPhotometer</td><td></td></tr><tr><td>Stand and clamp</td><td>Fisher Scientific</td><td>14-679Q and 05-769-8FQ</td><td>available from numerous suppliers</td></tr><tr><td>Sterile filter unit</td><td>Fisher Scientific (Nalgene)</td><td>09-740-65B</td><td>0.2 &amp;micro;m rapid-flow filter unit (150 mL)</td></tr><tr><td>Sterile filter unit 0.2 &amp;micro;m (syringe)</td><td>Fisher Scientific&amp;nbsp;</td><td>SLGV004SL</td><td>Millipore Sigma Milex 0.22 &amp;micro;m filter unit that attaches to syringe</td></tr><tr><td>Super speed centrifuge</td><td>Eppendorf&amp;nbsp;</td><td>5810R</td><td>with Eppendorf F34-6-38 fixed angle rotor (12,000 rpm)</td></tr><tr><td>Syringe (1 mL)</td><td>BD&amp;nbsp;</td><td>309628</td><td>1-mL syringe Luer-lok tip - sterile</td></tr><tr><td>Syringe (3 mL)</td><td>BD&amp;nbsp;</td><td>309656</td><td>3-mL syringe slip tip - sterile</td></tr><tr><td>Table-top centrifuge (low speed)</td><td>Eppendorf&amp;nbsp;</td><td>5702</td><td>with swinging bucket rotor</td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific</td><td>13-711-9AM</td><td>polyethylene 3.4 mL transfer pipette</td></tr><tr><td>Tris-base</td><td>Millipore Sigma</td><td>648310-M</td><td>Molecular Biology grade&amp;nbsp;</td></tr><tr><td>TrypLE select protease solution</td><td>Gibco (ThermoFisher)</td><td>12604013</td><td>TrypLE express enzyme (1x), no phenol red</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-80 XP</td><td>with Beckman SW41 rotor (41,000 rpm)</td></tr><tr><td>Vaporizer&amp;nbsp;</td><td>General Anesthetic Services, Inc.</td><td>Tec 3</td><td>Isoflurane vaporizer</td></tr><tr><td>Vortex Mixer</td><td>VWR</td><td>10153-838</td><td>analog vortex mixer</td></tr></tbody></table>

expression of transgenes in native bladder urothelium using adenovirus-mediated transduction

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense and transmit information about its environment to the underlying tissues, promoting voiding function and behavior. Disruption of the urothelial barrier, or its sensory/transducer function, can lead to disease. Studying these complex events is hampered by lack of simple strategies to alter gene and protein expression in the urothelium. Methods are described here that allow investigators to generate large amounts of high-titer adenovirus, which can then be used to transduce rodent urothelium with high efficiency, and in a relatively straightforward manner. Both cDNAs and small interfering RNAs can be expressed using adenoviral transduction, and the impact of transgene expression on urothelial function can be assessed 12 h to several days later. These methods have broad applicability to studies of normal and abnormal urothelial biology using mouse or rat animal models.

Virus preparation 
An example of virus purification by density gradient centrifugation is shown in Figure 1A. The light pink band, found at the interface of the loaded cellular material and the 1.25 g/mL CsCl layer, is primarily composed of disrupted cells and their debris (see magenta arrow in Figure 1A). It derives its pinkish color from the small amount of culture medium that is carried over from step 1.5 in the protocol. The virus parti...

Watch this Scientific Journal Video about Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction at JoVE.com

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

Methods are described for the generation of large amounts of recombinant adenoviruses, which can then be used to transduce the native rodent urothelium allowing for expression of transgenes or downregulation of endogenous gene products.

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense ...

expression-transgenes-native-bladder-urothelium-using-adenovirus

Research

JoVE Journal

Biology

1.4K Views.  University of Pittsburgh School of Medicine. Dit protocol beschrijft methoden waarmee onderzoekers CDNA's of SIRNA's kunnen uitdrukken in het inheemse blaasurotheel. De belangrijkste voordelen van deze techniek zijn de relatieve eenvoud, het vermogen om cDNA's, siRNA's in het urotheel met hoge efficiëntie en binnen een relatief korte tijd tot expressie te brengen. Het moeilijkste deel van deze techniek is de katheterisatie.De algehele techniek is echter relatief gemakkelijk onder de knie te krijgen als je het eenmaal hebt geleer...

Video: Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Medicine

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

Cancer Research

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

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

Source: Chuan, Y. et al. <a href="https://www.jove.com/video/57649" target="_blank">Novel method of plasmid DNA delivery to mouse bladder urothelium by electroporation.</a> J. Vis. Exp.&#160; (2018)
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.

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

Samenvatting

Meer video's verkennen