Name: novel method of plasmid dna delivery to mouse bladder urothelium by electroporation
Uploaded: 2018-05-03T14:30:00
Description: Watch this Scientific Journal Video about Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation at JoVE.com

Vi tackar Professor Bin Chen, Dr Yue Zhang och Kendy Hoang för hjälp med att ställa in elektroporation systemet. Detta arbete stöds av bidrag från Santa Cruz Cancer nytta Group och NIH till Z.A.W.

Alla förfaranden beskrivs här utfördes i enlighet med de riktlinjer och förordningar från institutionella djur vård och användning kommittén (IACUC) av University of California Santa Cruz.
1. plasmid och verktyg förberedelse
<ol><li>För varje urinblåsan till transfect, förbereda minst 20 µL av DNA plasmid (1 µg/µL).</li>
	<li>Tillsätt 1 µL av Trypan blå till de 20 µL av Plasmiden lösning i en 1,5 mL mikrocentrifug rör. Pipetten att blanda väl.</li>
	<li>Autoklav alla ki...

<ol>
	<li>Sharpless, N. E., Depinho, R. 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+mighty+mouse:+genetically+engineered+mouse+models+in+cancer+drug+development.">The mighty mouse: genetically engineered mouse models in cancer drug development.</a> Nat Rev Drug Discov. 5 (9), 741-754 (2006).</li><li>Heyer, J., Kwong, L. N., Lowe, S. W., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-germline+genetically+engineered+mouse+models+for+translational+cancer+research.">Non-germline genetically engineered mouse models for translational cancer research.</a> Nat Rev Cancer. 10 (7), 470-480 (2010).</li><li>Maddalo, D., 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+engineering+of+oncogenic+chromosomal+rearrangements+with+the+CRISPR/Cas9+system.">In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.</a> Nature. 516 (7531), 423-427 (2014).</li><li>Xue, W., 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=CRISPR-mediated+direct+mutation+of+cancer+genes+in+the+mouse+liver.">CRISPR-mediated direct mutation of cancer genes in the mouse liver.</a> Nature. , (2014).</li><li>Sanchez-Rivera, F. 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=Rapid+modelling+of+cooperating+genetic+events+in+cancer+through+somatic+genome+editing.">Rapid modelling of cooperating genetic events in cancer through somatic genome editing.</a> Nature. 516 (7531), 428-431 (2014).</li><li>Romero, 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=Keap1+loss+promotes+Kras-driven+lung+cancer+and+results+in+dependence+on+glutaminolysis.">Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.</a> Nat Med. 23 (11), 1362-1368 (2017).</li><li>Platt, R. 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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159 (2), 440-455 (2014).</li><li>Yin, H., 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=Genome+editing+with+Cas9+in+adult+mice+corrects+a+disease+mutation+and+phenotype.">Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.</a> Nat Biotechnol. 32 (6), 551-553 (2014).</li><li>Yang, Y., 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+dual+AAV+system+enables+the+Cas9-mediated+correction+of+a+metabolic+liver+disease+in+newborn+mice.">A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.</a> Nat Biotechnol. 34 (3), 334-338 (2016).</li><li>Negrete, H. O., Lavelle, J. P., Berg, J., Lewis, S. A., Zeidel, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+properties+of+the+intact+mammalian+bladder+epithelium.">Permeability properties of the intact mammalian bladder epithelium.</a> Am J Physiol. 271 (4 Pt 2), F886-F894 (1996).</li><li>Lilly, J. D., Parsons, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+surface+glycosaminoglycans+is+a+human+epithelial+permeability+barrier.">Bladder surface glycosaminoglycans is a human epithelial permeability barrier.</a> Surg Gynecol Obstet. 171 (6), 493-496 (1990).</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> Mol Ther. 10 (4), 697-705 (2004).</li><li>Yamashita, 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=Syn3+provides+high+levels+of+intravesical+adenoviral-mediated+gene+transfer+for+gene+therapy+of+genetically+altered+urothelium+and+superficial+bladder+cancer.">Syn3 provides high levels of intravesical adenoviral-mediated gene transfer for gene therapy of genetically altered urothelium and superficial bladder cancer.</a> Cancer Gene Ther. 9 (8), 687-691 (2002).</li><li>Engler, H., 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=Ethanol+improves+adenovirus-mediated+gene+transfer+and+expression+to+the+bladder+epithelium+of+rodents.">Ethanol improves adenovirus-mediated gene transfer and expression to the bladder epithelium of rodents.</a> Urology. 53 (5), 1049-1053 (1999).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> P Natl Acad Sci USA. 101 (1), 16-22 (2004).</li><li>Yarmush, M. L., Golberg, A., Sersa, G., Kotnik, T., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+technologies+for+medicine:+principles,+applications,+and+challenges.">Electroporation-based technologies for medicine: principles, applications, and challenges.</a> Annu Rev Biomed Eng. 16, 295-320 (2014).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li><li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3 (4), e1883 (2008).</li><li>Saito, T. <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+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protoc. 1 (3), 1552-1558 (2006).</li><li>Molotkov, D. A., Yukin, A. Y., Afzalov, R. A., Khiroug, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+postnatal+rat+brain+by+non-ventricular+plasmid+injection+and+electroporation.">Gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation.</a> J Vis Exp. (43), (2010).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. (6), e239 (2007).</li><li>Follenzi, A., Santambrogio, L., Annoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+lentiviral+vectors.">Immune responses to lentiviral vectors.</a> Curr Gene Ther. 7 (5), 306-315 (2007).</li></ol>

Elektroporation ökar permeabilitet cellmembranet och är en kraftfull teknik för gen electrotransfer16. Gen leverans till levande gnagare av elektroporation har ofta använts i neurobiologi fält17,18,19,20,21. Dess potentiella tillämpningar i många andra organ har inte undersökts utförligt. Till vår kunskap är vår metod som be...

Genetiskt modifierade musmodeller (GEMMs) har spelat en viktig roll i att utöka vår kunskap om djurs utveckling, gen funktioner i vivooch mekanismer för sjukdomsprogression1,2. Könsceller GEMMs utvecklas traditionellt på två sätt. Först är skapandet av transgena möss genom pronukleär injektion av DNA plasmid följt av transplantation av zygoter i pseudopregnant honor. Den andra är generationen av knock-out/inpressning möss av homolog rekombination i embryonala stamceller och utvecklingen av chimärer. Villkorliga knockout av gener i särskilda vävnads- eller typ av intresse innebär uppfödning av genmanipulerade alleler med Cre och flox platser för flera generationer. Hela processen kan vara dyrt och tidskrävande, särskilt om målet är att rikta flera gener vävnad-specifikt. Nyligen, genredigering teknik såsom klustrade regelbundet mellanliggande kort palindromic repetitioner (CRISPR) har tillämpats på flera organ av mössen att inducera vävnadsspecifika genetiska förändringar för autoktona cancer modellering3, 4,5,6,7 eller rätt sjukdom mutationer i situ8,9. I dessa studier var leveransen av gärna vektorerna vanligtvis uppnås genom adenovirala eller lentiviral infektion i orgel eller hydrodynamiska svans-ven injektion. Den relativa lätthet leverans av CRISPR komponenterna till lunga och lever har förbättrats avsevärt bekvämlighet och effektivitet av cancer modellering i dessa organ jämfört med traditionella Cre-Lox baserat musmodeller.
Det blåsan urotelet är där de flesta urinblåsan tumörer har sitt ursprung. Leverans av DNA vektorer till det blåsan urotelet för cancerbehandling modellering eller genen hämmas av ett glykosaminoglykan lager på den apikala urothelial ytan, som fungerar som en barriär för efterlevnad av infektiösa virus10,11. Flera förbehandling medel såsom Syn3 och natriumdodecylsulfat-β-d-maltoside har använts för att störa denna barriär och förbättra adenovirus infektion effektivitet12,13,14. Om adeno-associerade virus (AAVs) effektivt kan infektera blåsan har inte rapporterats. Sammantaget vore en icke-virala baserat leveransmetod önskvärt. Här beskriver vi en ny metod som utvecklats i laboratoriet för effektiv DNA plasmid leverans till mus urotel genom urinröret kateterisering och elektroporation. Eftersom vår metod inte innebär kemisk förbehandling av glykosaminoglykan lagret eller virus produktion, det avsevärt förenklar distribution av DNA plasmider i mus urinblåsan urotel och bör underlätta olika in-vivo studier av urinblåsan sjukdomar.

<table>
	<tbody>
		<tr>
			<td>BD 1mL TB Syringe</td>
			<td>Fisher</td>
			<td>148232E</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Sigma</td>
			<td>B9275-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-5005</td>
			<td>Treat with 70% ethanol before surgical use</td>
		</tr>
		<tr>
			<td>ECM830 SQ Wave Electroporator</td>
			<td>Harvard Apparatus</td>
			<td>450052</td>
			<td></td>
		</tr>
		<tr>
			<td>Exel International Disposable Safelet I.V. Catheters</td>
			<td>Fisher Scientific</td>
			<td>14-841-21</td>
			<td>24G, 2.11 cm length, 0.045 cm outer diameter</td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-5135</td>
			<td>Treat with 70% ethanol before surgical use</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vet one</td>
			<td>501017</td>
			<td></td>
		</tr>
		<tr>
			<td>K&#38;H Heated Resting Mat for Small Animals, 9 By 12 Inches</td>
			<td>Amazon</td>
			<td>n/a</td>
			<td>Cover the heat mat with paper towels</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Fisher</td>
			<td>NC0849514</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH7.4</td>
			<td>Life Technologies</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips 200 &#181;L</td>
			<td>USA Scientific</td>
			<td>1111-0206</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Tips, Universal Fit; 0.1 to 10 &#181;L</td>
			<td>Fisher</td>
			<td>02707438(CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPETMAN NEO P2</td>
			<td>Gilson</td>
			<td>F144561</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPETMAN NEO P200N</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid CAG-GFP</td>
			<td>Addgene</td>
			<td>16664</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum tweezertrode 5 mm</td>
			<td>Harvard Apparatus</td>
			<td>450489</td>
			<td>5 mm diameter</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-5880</td>
			<td>Treat with 70% ethanol before surgical use</td>
		</tr>
		<tr>
			<td>String cord</td>
			<td>Amazon</td>
			<td>n/a</td>
			<td>Mandala Crafts 150D 210D 0.8mm 1mm leather sewing stitching flat waxed thread string cord</td>
		</tr>
		<tr>
			<td>Tape, Scotch</td>
			<td>Fisher</td>
			<td>19047257</td>
			<td></td>
		</tr>
		<tr>
			<td>Therio-gel Veterinary Lubricant</td>
			<td>PBS animal health</td>
			<td>353-736</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 Table top system anesthesia</td>
			<td>VetEquip</td>
			<td>901806</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl violet suture 18&#34; FS-2 cutting 5-0</td>
			<td>Fisher</td>
			<td>NC0578475</td>
			<td></td>
		</tr>
		<tr>
			<td>Walgreens Povidone Iodine 10%</td>
			<td>Walgreens</td>
			<td>953982</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clip</td>
			<td>Fisher</td>
			<td>018045</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clip applier</td>
			<td>Fisher</td>
			<td>01804</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel method of plasmid dna delivery to mouse bladder urothelium by electroporation

Genetiskt modifierade musmodeller (GEMMs) är extremt värdefulla i avslöjande romanen biologiska insikter i initiering och progression mekanismer för mänskliga sjukdomar såsom cancer. Transgena och villkorliga knockoutmöss har ofta använts för genen överuttryck eller ablation i specifika vävnader eller cell typer i vivo. Däremot kan genererar könsceller musmodeller vara tidskrävande och kostsamma. Senaste framsteg i gen redigeringen och genomförbarheten av att leverera DNA plasmider av virusinfektion har aktiverat snabb generering av icke-könsceller autoktont cancer musmodeller för flera organ. Blåsan är ett organ som har varit svårt för virala vektorer tillgång till, på grund av närvaron av en glykosaminoglykan lager som täcker urotelet. Här beskriver vi en ny metod som utvecklades i lab för effektiv leverans av DNA plasmider in mus urinblåsan urotel i vivo. Genom intravesical instillation av pCAG-GFP DNA plasmid och elektroporation av kirurgiskt exponerade urinblåsan visar vi att DNA Plasmiden kan levereras specifikt in i urinblåsan urothelial celler för övergående uttryck. Vår metod ger ett snabbt och bekvämt sätt för överuttryck och Nedslagning av gener i mus urinblåsan, och kan användas till att bygga GEMMs av cancer i urinblåsan och andra urologiska sjukdomar.

För att demonstrera framgång leveranssätt genen in i urinblåsan urotel, använde vi pCAG-GFP15 plasmiden för elektroporation. 20 µL av plasmid och Trypan blå lösningen injicerades genom urinröret och 5 elektriska pulser administrerades. Efter 48 h, vi dissekerade mus blåsan och observerade fläckar av GFP fluorescens i dissektion fluorescens Mikroskop, medan en negativ kontrollerar urinblåsan som inte genomgick elektroporation visade ingen GFP signal (<s...

Watch this Scientific Journal Video about Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation at JoVE.com

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

Vi beskriver en ny metod för leverans av DNA plasmid i urothelial celler mus blåsan i vivo genom urinröret kateterisering och elektroporation. Det erbjuder ett snabbt och bekvämt sätt för att generera autoktont musmodeller av urinblåsan sjukdomar.

Romanen leveranssätt Plasmid DNA till mus urinblåsan urotel av elektroporation

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

The overall goal of this method is to deliver DNA plasmids into the bladder urothelium of live mice in vivo for gene overexpression or genome editing. This method can help answer key questions in the bladder cancer field, such as how do different genetic mutations promote bladder cancer progression? The main advantage of this technique is that it provides a fast and convenient way for delivering genes into bladder without the safety concerns and technical barriers associated with viral vectors.I first had the idea for this method of trying to use any engineered associated viruses for DNA delivery, which didn't work well the bladder. The implication of this technique can also extend towards gene therapy because electroporation can achieve efficient delivery of DNA plasmids into the urothelial cells. Demonstrating the procedure will be Ofir Stefanson and Yueli Liu from my laboratory.To begin this procedure, autoclave all surgical instruments and sterilize the workspace with 70%ethanol. Spray surgical instruments and the gloves with 70%ethanol frequently when performing the subsequent procedures to maintain sterile conditions. To begin, anesthetize the mouse and shave the surgical area.To deplete the urine, apply lubricant to the catheter and ensure that its outer surface is fully covered. Next, insert the catheter into the urethra opening of the mouse and slowly push it until it reaches the bladder. Gently press the abdomen to help urine depletion.Subsequently, discard the urine with a pipette into a waste beaker, then pipette 80 microliters of PBS into the outer end of the catheter with the other end still in the bladder. Next, carefully attach a one millimeter syringe to the catheter. Gently push the syringe to inject PBS into the bladder.Leave some PBS in the catheter to avoid creating air bubbles in the bladder. Afterward, remove the syringe and wait for PBS to drain out of the bladder. Gently press the abdomen to evacuate PBS, then discard the PBS in the catheter with a pipette into the waste beaker.Repeat PBS washing for two more times. For each bladder to transfect, prepare at least 20 microliters of DNA plasmid at one microgram per microliter. Next, add one microliter of trypan blue to the 20 microliters of plasmid solution and a 1.5 milliliter microcentrifuge tube and pipette to mix well.To deliver the plasmid, apply 70%ethanol to the abdomen. Make a vertical incision of one centimeter to open the abdominal skin above the bladder. Next, cut the muscle layer to expose the bladder.Pipette at least 20 microliters of plasmid plus trypan blue solution into the outer end of the catheter and detach it to the syringe. Then, inject the plasmid solution into the bladder. If the bladder turns blue, the injection is successful.Leave some liquid in the catheter to avoid creating air bubbles in the bladder. Afterward, grab the external urethral oraifice and remove the catheter and syringe. Use a string to tighten the external urethral orifice.Make two loops with the string, and then tie with two knots. This is a crucial step that requires two people to cooperate. Make sure the knots are tight so the plasmids will not leak out.Now, turn on the electroporation generator. Pinch the bladder with two electrodes and push the foot pedal to perform electroporation. To keep the bladder propped one can pinch the back of the abdominal incision with tweezers.Avoid any contact between tweezers and the electrodes as this may generate a spark. When it is done, suture the abdominal and skin opening with sterile, surgical suture. Remove the string and apply clips.Apply iodine onto the wound, and then remove the animal from the isoflurane system. Administer buprenorphine analgesic subcutaneously to alleviate post surgical pain. Keep the animal on the heating pad and return it to the home cage after full recovery.Check the animal at least once daily for normal mobility, drinking and feeding behaviors, and no signs of infection until the incision is healed. Then, a week later, remove the clips. Administer subsequent injections of buprenorphine analgesic if the animal displays pain symptoms such as reduced grooming, increased aggressiveness, and vocalization.To demonstrate successful gene delivery into the bladder urothelium, 20 microliters of pCAG-GFP plasmid and trypan blue solution was injected through the urethra and five electric pulses were administered. Shown here is the successful electroporation that turned the bladder green after 48 hours, whereas a negative control bladder that did not undergo electroporation showed no GFP signal. Immunofluorescent staining of sectioned bladder tissues was performed with CK5, CK18, and GFP antibodies.GFP signal was observed to be present in umbrella, intermediate, and basal cells, but not at the muscle layer indicating good specificity of plasmid delivery into the urothelium. Once mastered, this technique can be performed in 15 minutes if performed properly. After its development, this technique paves the way for researchers in the field of bladder cancer to build new autochthonous mouse models to study disease progression mechanisms.

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DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

DNA Transfektion av däggdjurs Skelettmuskulatur hjälp In Vivo Elektroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

Primär Kultur och Plasmid elektroporation av Murine organ Corti.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

Strömlinjeformad Rening av Plasmid DNA från Prokaryota kulturer

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Direkt Leverans av MIF Morpholinos Into the Zebrafish Otocyst med injektion och elektroporation Påverkar Inner Ear utveckling

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

En praktisk och ny metod att extrahera genomisk DNA från Kits blodinsamling för Plasma Protein Preservation

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel<em&gt; Ex vivo</em&gt; Kultur Metod för embryonala Mouse Heart

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Spårning av Hematopoietic Stem och stamceller i vuxen mus calvarial Benmärg

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

Plasmid-härledda DNA Strand Displacement Gates för genomförande Kemisk reaktions Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...