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>

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160; (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 &#8805; 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 &#8805; 99%</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</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 &#8805; 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&#160; 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 &#8805; 99%</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Millipore Sigma</td>
			<td>P5655</td>
			<td>Cell culture grade&#160; &#8805; 99%</td>
		</tr>
		<tr>
			<td>Na2HPO4&#8226;7 H2O</td>
			<td>Millipore Sigma</td>
			<td>431478</td>
			<td>&#160;&#8805; 99.99%</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Millipore Sigma</td>
			<td>S3014</td>
			<td>Molecular Biology grade &#8805; 99%</td>
		</tr>
		<tr>
			<td>N-dodecyl-&#946;-D-maltoside&#160;</td>
			<td>Millipore Sigma</td>
			<td>D4641</td>
			<td>&#160;&#8805; 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&#160;</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&#160;</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 &#181;m rapid-flow filter unit (150 mL)</td>
		</tr>
		<tr>
			<td>Sterile filter unit 0.2 &#181;m (syringe)</td>
			<td>Fisher Scientific&#160;</td>
			<td>SLGV004SL</td>
			<td>Millipore Sigma Milex 0.22 &#181;m filter unit that attaches to syringe</td>
		</tr>
		<tr>
			<td>Super speed centrifuge</td>
			<td>Eppendorf&#160;</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&#160;</td>
			<td>309628</td>
			<td>1-mL syringe Luer-lok tip - sterile</td>
		</tr>
		<tr>
			<td>Syringe (3 mL)</td>
			<td>BD&#160;</td>
			<td>309656</td>
			<td>3-mL syringe slip tip - sterile</td>
		</tr>
		<tr>
			<td>Table-top centrifuge (low speed)</td>
			<td>Eppendorf&#160;</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&#160;</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&#160;</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

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1.4K Views.  University of Pittsburgh School of Medicine. 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.

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

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 ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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 ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Methods Article

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