A Career Development Award from the Department of Veterans Affairs [BX002797] supported L.E.W. and the National Institutes of Health [R01-DK095867] and American Heart Association [15GRNT25700209] supported J.C. The National Institutes of Health [DK093660], Department of Veterans Affairs [BX002190], and the Vanderbilt Center for Kidney Disease supported M.H.W. This material is the result of work supported with resources and use of facilities at the VA Tennessee Valley Healthcare System.

All methods described here have been approved by the Institutional Animal Care and Use Committees (IACUCs) of Baylor College of Medicine and Vanderbilt University Medical Center.
1. Prepare the DNA solution for injection
<ol>
	<li>Select the plasmid(s) to express the transgene(s) carefully to maximize the desirable characteristics to improve transfection efficiency and transgene expression. 
	NOTE: Hydrodynamic renal pelvis injection of plasmids to express the fluorescent marker TdTomat...

<ol>
	<li>Liu, F., Song, Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics-based+transfection+in+animals+by+systemic+administration+of+plasmid+DNA.">Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.</a> Gene Ther. 6 (7), 1258-1266 (1999).</li><li>Zhang, G., Budker, V., Wolff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+foreign+gene+expression+in+hepatocytes+after+tail+vein+injections+of+naked+plasmid+DNA.">High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.</a> Human Gene Therapy. 10, 1735-1737 (1999).</li><li>Fumoto, S., Nishimura, K., Nishida, K., Kawakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+of+the+Intracellular+Fate+of+Plasmid+DNA+and+Transgene+Expression:+ZsGreen1+and+Tissue+Clearing+Method+CUBIC+Are+an+Optimal+Combination+for+Multicolor+Deep+Imaging+in+Murine+Tissues.">Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.</a> PLoS One. 11 (1), e0148233 (2016).</li><li>Yoshino, H., Hashizume, K., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+plasmid+DNA+transfer+to+the+porcine+liver+using+rapid+injection+with+large+volume.">Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume.</a> Gene Ther. 13 (24), 1696-1702 (2006).</li><li>Kamimura, K., Suda, T., Xu, W., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,+lobe-specific+hydrodynamic+gene+delivery+to+swine+liver.">Image-guided, lobe-specific hydrodynamic gene delivery to swine liver.</a> Mol Ther. 17 (3), 491-499 (2009).</li><li>Kamimura, K., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,intravascular+hydrodynamic+gene+delivery+to+skeletal+muscle+in+pigs.">Image-guided,intravascular hydrodynamic gene delivery to skeletal muscle in pigs.</a> Mol Ther. 18 (1), 93-100 (2010).</li><li>Suda, T., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+gene+delivery:+its+principles+and+applications.">Hydrodynamic gene delivery: its principles and applications.</a> Mol.Ther. 15 (12), 2063-2069 (2007).</li><li>Alino, S. 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=Naked+DNA+delivery+to+whole+pig+cardiac+tissue+by+coronary+sinus+retrograde+injection+employing+non-invasive+catheterization.">Naked DNA delivery to whole pig cardiac tissue by coronary sinus retrograde injection employing non-invasive catheterization.</a> J Gene Med. 12 (11), 920-926 (2010).</li><li>Suda, T., Gao, X., Stolz, D. B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14 (2), 129-137 (2007).</li><li>Skrypnyk, N. I., Harris, R. C., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+model+of+acute+kidney+injury+and+post+injury+fibrosis+in+mice.">Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice.</a> J Vis Exp. (78), (2013).</li><li>Woodard, L. E., 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=Kidney-specific+transposon-mediated+gene+transfer+in+vivo.">Kidney-specific transposon-mediated gene transfer in vivo.</a> Sci Rep. 7, 44904 (2017).</li><li>Liang, A., 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=Loss+of+glutathione+S-transferase+A4+accelerates+obstruction-induced+tubule+damage+and+renal+fibrosis.">Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis.</a> Journal of Pathology. 228 (4), 448-458 (2012).</li><li>Liang, 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=Protective+role+of+insulin-like+growth+factor-1+receptor+in+endothelial+cells+against+unilateral+ureteral+obstruction-induced+renal+fibrosis.">Protective role of insulin-like growth factor-1 receptor in endothelial cells against unilateral ureteral obstruction-induced renal fibrosis.</a> Am J Pathol. 185 (5), 1234-1250 (2015).</li><li>Corridon, P. R., 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+method+to+facilitate+and+monitor+expression+of+exogenous+genes+in+the+rat+kidney+using+plasmid+and+viral+vectors.">A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors.</a> Am J Physiol Renal Physiol. 304 (9), F1217-F1229 (2013).</li><li>Wallace, D. 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=Tracking+kidney+volume+in+mice+with+polycystic+kidney+disease+by+magnetic+resonance+imaging.">Tracking kidney volume in mice with polycystic kidney disease by magnetic resonance imaging.</a> Kidney Int. 73 (6), 778-781 (2008).</li><li>Woodard, L. E., Wilson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-ing+models+and+new+therapeutic+strategies.">piggyBac-ing models and new therapeutic strategies.</a> Trends Biotechnol. 33 (9), 525-533 (2015).</li><li>Yeikilis, R., 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=Hydrodynamics+based+transfection+in+normal+and+fibrotic+rats.">Hydrodynamics based transfection in normal and fibrotic rats.</a> World J Gastroenterol. 12 (38), 6149-6155 (2006).</li><li>Thalhofer, C. 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=In+vivo+imaging+of+transgenic+Leishmania+parasites+in+a+live+host.">In vivo imaging of transgenic Leishmania parasites in a live host.</a> J Vis Exp. (41), (2010).</li><li>Wooddell, C. 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=Muscle+damage+after+delivery+of+naked+plasmid+DNA+into+skeletal+muscles+is+batch+dependent.">Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.</a> Hum Gene Ther. 22 (2), 225-235 (2011).</li><li>Crespo, A., 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=Hydrodynamic+liver+gene+transfer+mechanism+involves+transient+sinusoidal+blood+stasis+and+massive+hepatocyte+endocytic+vesicles.">Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.</a> Gene Ther. 12 (11), 927-935 (2005).</li><li>Rocca, C. J., Ur, S. N., Harrison, F., Cherqui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rAAV9+combined+with+renal+vein+injection+is+optimal+for+kidney-targeted+gene+delivery:+conclusion+of+a+comparative+study.">rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.</a> Gene Ther. 21 (6), 618-628 (2014).</li></ol>

In this protocol a robust method for achieving reproducible gene expression specifically in the kidney is described. In the hands of a moderately experienced surgeon we have found the percentage of mice transfected by this technique to be in the range of 50-100%, depending on mouse age and the sensitivity of the readout of the transgene. The level of luciferase gene expression was above background for several months in mice receiving piggyBac transposons and completely maintained for several weeks in immunocompr...

The hydrodynamic tail vein injection technique has become a commonly used way to achieve high levels of gene expression in mouse liver1,2. The kidneys are also transfected by this technique at a much lower level, approximately 100-fold less3. The hydrodynamic renal pelvis injection described here provides a simple way to control the specificity of organ expression through physical means using the same hydrodynamic principles that have been established previously in liver4,5, muscle6, and other organs7,8. This method transfects cells in live animals in vivo by using pressure and speed to force fluid containing DNA into the cells, simultaneously inducing damage to the organ that is quickly resolved9. Using well-established surgical techniques to visualize the kidney via a flank incision10 along with a single injection by insulin syringe, we have found successful transfection of various types of kidney cells, mainly interstitial fibroblasts, tubules, and collecting duct11. Dissection of these mice has shown that other organs are not transfected at levels high enough to visualize by luciferase imaging techniques11. Since the technique is non-viral, use of plasmid DNA for transfection permits fast and easy preparation of the reagents required for injection.
We have used localized hydrodynamic injections to express the antioxidant glutathione S-transferase A4, the insulin-like growth factor-1 receptor, and the hormone erythropoietin in the kidney, all with the expected biological effects11,12,13. Detailed evaluation of route of administration, injection volume, DNA dosage, and promoter choice has been performed11. Additionally, both the piggyBac transposon system and/or cyclophosphamide treatment to suppress the immune reaction to the transgene have been shown to improve long-term gene expression outcomes11. Other investigators have used a renal vein approach in rat with success, achieving high transfection efficiency for time periods of greater than one month14. However, genetic correction of phenotypes mimicking human disease are usually performed in mice first as a proof-of-concept since most mammalian genetic models are mouse models. We compared renal vein injection to renal pelvis injection and found that injection into the renal pelvis was superior to the renal vein for gene expression (approximately ten-fold higher) and survival11. The renal pelvis is an ideal route of entry into the kidney because it is flexible enough to tolerate fluctuations in urine production and is often able to maintain its structural integrity even when dilated during hydronephrosis. Additionally, injection into the renal pelvis allowed access to the kidney without piercing the kidney capsule, allowing the injected fluid to be visibly retained by the kidney better than intraparenchymal injection. Other mouse organs do not have a route of entry other than the vasculature, but the urinary space of the kidney is an ideal injection site. Additionally, injection into the renal vein resulted in leakage of blood into the abdominal cavity. The total kidney volume of wild-type mouse kidneys has been estimated by magnetic resonance imaging to be approximately 0.2 cm3, so the volume of a single kidney is approximately equal to the amount of fluid injected by renal pelvis hydrodynamic injection (100 &#181;L)15. Herein, we have made available all of the detailed nuances of the hydrodynamic renal pelvis injection protocol to achieve reproducible transfection of the kidney.

<table>
	<tbody>
		<tr>
			<td>AnaSed Xylazine</td>
			<td>Patterson Veterinary</td>
			<td>07-808-1947</td>
			<td>Anesthetic - Not controlled substance</td>
		</tr>
		<tr>
			<td>BD Insulin Syringe 0.5 mL 29G 1/2 Inch</td>
			<td>Cardinal Health</td>
			<td>309306</td>
			<td>Required syringes</td>
		</tr>
		<tr>
			<td>Buprenex</td>
			<td>Pharmacist/Veterinarian</td>
			<td></td>
			<td>Analgesia - Controlled Substance</td>
		</tr>
		<tr>
			<td>Dynarex Disposable Towel Drape</td>
			<td>Thermo Fisher Scientific</td>
			<td>19-310-671</td>
			<td>Place over heat pad</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>Use only endotoxin-free plasmid DNA</td>
		</tr>
		<tr>
			<td>Endosafe Gel-Clot LAL Rapid Positive Control</td>
			<td>Charles River</td>
			<td>PC200</td>
			<td>Positive control for endotoxin test</td>
		</tr>
		<tr>
			<td>Endosafe Gel-Clot LAL Rapid Single Test Vial</td>
			<td>Charles River</td>
			<td>R13500</td>
			<td>Endotoxin test</td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5882</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Fisherbrand Instant Sealing Sterilization Pouch - 9&#34;</td>
			<td>Thermo Fisher Scientific</td>
			<td>01-812-51</td>
			<td>For autoclaving surgical tools</td>
		</tr>
		<tr>
			<td>Gaymar Heat Pump</td>
			<td>Paragon Medical</td>
			<td>TP-700</td>
			<td>Water-circulating heat pump</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Roboz Surgical Instrument</td>
			<td>DS-401</td>
			<td>To reuse surgical tools during surgery</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5136</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Graefe Tissue Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5153</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Halsey Needle Holder, 5&#34; Length</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-7841</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Heat pads - 15&#34; x 21&#34; - need at least 3</td>
			<td>Paragon Medical</td>
			<td>TP22G</td>
			<td>For use with Gaymar Heat Pump</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane, USP)</td>
			<td>Abbott Animal Health</td>
			<td>5260-04-05</td>
			<td>For imaging and euthanasia</td>
		</tr>
		<tr>
			<td>Isotec Isoflurane Delivery System Vaporizor</td>
			<td>Smiths Medical</td>
			<td>VCT3K2</td>
			<td>For imaging and euthanasia</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pharmacist/Veterinarian</td>
			<td></td>
			<td>Anesthetic - Controlled Substance</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34120</td>
			<td>Laboratory tissues</td>
		</tr>
		<tr>
			<td>Living Image software</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td>For live animal imaging</td>
		</tr>
		<tr>
			<td>Luciferin</td>
			<td>Perkin Elmer</td>
			<td>122796</td>
			<td>For live animal imaging</td>
		</tr>
		<tr>
			<td>Nanodrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000-US-CAN</td>
			<td>Spectrophotometer for DNA measurement</td>
		</tr>
		<tr>
			<td>Prevantics Swabs</td>
			<td>Thermo Fisher Scientific</td>
			<td>23-100-110</td>
			<td>For skin surgery prep</td>
		</tr>
		<tr>
			<td>Prolene 5-0 sutures Taper 30&#34;</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC0256891</td>
			<td>Non-absorbable sutures for skin</td>
		</tr>
		<tr>
			<td>Puralube Brand Opthalmic Ointment</td>
			<td>Patterson Veterinary</td>
			<td>07-888-2572</td>
			<td>To keep eyes moist during surgery</td>
		</tr>
		<tr>
			<td>Trans IT - QR Hydrodynamic Delivery Solution</td>
			<td>Mirus Bio</td>
			<td>MIR-5240</td>
			<td>Hydrodynamic delivery buffer for diluting DNA</td>
		</tr>
		<tr>
			<td>Vicryl 5-0 Sutures J303H</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9816710</td>
			<td>Absorbable sutures for muscle layer</td>
		</tr>
		<tr>
			<td>Wahl Mini Arco Clipper</td>
			<td>Med-Vet International</td>
			<td>8787-1550</td>
			<td>Shaver for skin prep</td>
		</tr>
		<tr>
			<td>Xenogen IVIS 200</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td>For live animal imaging</td>
		</tr>
	</tbody>
</table>

hydrodynamic renal pelvis injection for non-viral expression of proteins in the kidney

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.

The surgery and injection technique are simple to perform once mastered, requiring no major equipment or expensive materials. If new to flank-incision kidney surgery, one training day on several mice scheduled for euthanasia should be allowed in which the mice are not recovered following surgery because the first attempt at this surgery may take much longer than normal. Alternatively, investigators familiar with similar techniques may find it quite simple. Carefully follow the illustratio...

Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney.

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

The overall goal of this surgical procedure is to produce protein expression from plasmid DNA in the adult mouse kidney. This method is a new tool to address key questions in nephrology, such as, how the expression of a protein of interest affects the adult kidney. The main advantage of this technique is that it produces protein expression in a reproducible way, specifically in the kidney, unlike other methods that may also produce gene expression in other organs.Visual demonstration of this method is critical as injection of the renal pelvis requires careful exposure and must be done quickly in order for the plasma DNA to enter the cells. Start by resuspending plasmid DNA in the hydrodynamic delivery buffer. On a water circulated heat pad covered with a blue pad, place the mouse.Using a shaver designed for pet grooming, shave the left side of the back of the mouse from shoulder to rump and flank to spine. Clean loose hair and debris and apply vet ointment in both it's eyes to prevent corneal desiccation. Now, prepare 100 microliters containing 10 to 50 micrograms diluted plasmid DNA in a separate syringe for each mouse that is anesthetized, ensuring that there are no air bubbles.The needle should be permanently attached without a safety. Prior to performing the surgery, empty sterile surgical tools without touching onto a sterile tray placed on the heat pad. Now, pick up the mouse and lace it in an illuminated field of view.From the packing, remove 3.15%chlorhexidine gluconate and 70%isopropyl alcohol skin antiseptic swabs and place near the animal. Wear sterile surgical gloves and starting from the site of incision, swab the animal with a new chlorhexidine alcohol swab, three times in a circular motion. Now, locate the incision site.Pinch the skin with tweezers and using scissors, cut the skin layer approximately one centimeter from the spine, below the ribcage. Make another one centimeter cut below in the muscle layer. Locate the kidney and without touching it, gently expose it from the abdominal cavity by applying a steady gently pressure on the abdomen with the fingers.Exposing the kidney is sometimes difficult. Apply gentle pressure to the abdomen and spine to avoid damaging the spleen. If organs other than the kidney come out, gently return them to the body cavity using closed forceps if necessary.Then, gently separate the kidney from the surrounding fat just enough to visualize the renal pelvis, which can be seen as a small white dot. After visualizing the renal pelvis, use closed forceps to push down on the right side of the kidney so that the renal pelvis remains in view. Then, grasp the loaded insulin syringe in the right hand by holding it parallel to the working surface and insert the needle carefully into the renal pelvis.Once the needle is inserted, inject the solution quickly, within three seconds. Let the needle remain in place for approximately 10 seconds to prevent backflow of the solution. Then, remove the needle slowly and carefully and return the organ to the abdominal cavity by stretching the incision site gently using closed forceps.Now, suture the muscle layer with five-o absorbable sutures and place two to four independent knots. Similarly, suture the skin layer with five-O or six-O non-absorbable nylon sutures and place two to four independent knots. To assess transfection efficiency, introduce 10 microgram of a luciferase expressing plasmid to the diluted DNA for injection to perform live animal imaging, the following day.Place the first cage of mice into the chamber and seal it shut. Once anesthetized, inject each mouse intra-peritoneally with 100 microliters of thawed luciferin and record the time. Select the correct user ID and from the top menu bar, click on acquisition and select auto save two from the drop down menu.Initialize the system by clicking on initialize. Wait for the initializing to finish and change the field of view to D, to image five mice. Ensure that the animals are placed in the desired order and that they're backs are facing the camera so as to visualize the kidney.Then, press acquire five minutes following injection to take the image. After acquisition, remove the mice from the imaging machine. Turn off the gas.Clean the chamber, nose combs and the imaging stage. To analyze the data, after clicking on ROI tools, click the circle icon and select one on the drop down menu to place new ROI in the image as indicated by a red oval with four squares around it. To adjust the size of the ROI, place the cursor over a red square and drag to enclose the purple area overlaying the injected kidney.Now, right click this ROI and select duplicate ROI to duplicate the ROI in the current window and move it over to the next mouse. After placing all ROI's from the menu directly above the image, change the units from counts to radiance photons. Finally, create a spreadsheet of the measurements by exporting the ROI data from the tool palate by clicking on ROI tools and then on the pencil ruler icon.The kidney's specific gene expression of reporter plasmids is shown. All mice were injected with pTet plasmid expressing enhanced firefly luciferase, while mice two, three and five were also injected with pEF1-alpha-tdtomato plasmid, expressing the red fluorescent protein, tdtomato. Generally, the radiance in young mice will be within one order of magnitude.It is possible that some mice do not show appreciable levels of radiance, either due to low transfection efficiency or lack of the transgene itself. Injection of hydrodynamic buffer serves as a control. No plasmid expression is observed when given only the hydrodynamic buffer.In a first few days following transfection, there will likely be a low number of cells staining positive for the transgene. These cells are seen localized in patches in the areas infiltrated by the injection fluid. In this image, the green signals are from the proximal tubules, which are stained for lotus tetragonolobus agglutinin and the red signals are from the transfected cells expressing the tdtomato reporter gene.Once mastered, the surgery can be done in less than 10 minutes, however, the anesthesia takes time to induce and recover from. So, two trained personnel, working closely together would be able to do only about 10 to 15 mice per day. While attempting this procedure, it's important to remember to use young mice.The hydrodynamic technique does not work as well in older fibrotic organs. Following this procedure, other methods can be performed, such as analysis of blood, urine and kidney tissue samples, in order to assess the expression of the gene of interest. After watching this video, you should have a good understanding of how to inject the renal pelvis with plasmid DNA to achieve kidney specific protein expression.

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18.1K visualizações.  Tennessee Valley Healthcare System. The overall goal of this surgical procedure is to produce protein expression from plasmid DNA in the adult mouse kidney. This method is a new tool to address key questions in nephrology, such as, how the expression of a protein of interest affects the adult kidney. The main advantage of this technique is that it produces protein expression in a reproducible way, specifically in the kidney, unlike other methods that may also produce gene expression in other organs.Visual demonstration o...