Wir danken Professor Bin Chen, Dr. Yue Zhang und Kendy Hoang für Hilfe beim Einrichten der Elektroporation-System. Diese Arbeit wurde unterstützt durch Zuschüsse aus dem Santa Cruz Cancer nutzen Group und NIH, Z.A.W.

Alle hier beschriebene Verfahren wurden gemäß den Richtlinien und Verordnungen aus dem institutionellen Animal Care und Nutzung Committee (IACUC) der University of California Santa Cruz durchgeführt.
(1) Plasmid und Vorbereitung der Werkzeuge
<ol><li>Für jede Blase zu transfizieren, mindestens 20 µL DNA Plasmid (1 µg/µL) vorzubereiten.</li>
	<li>Die 20 µL Plasmid-Lösung in einem 1,5 mL Microcentrifuge Schlauch 1 µL Trypan blau hinzufügen. Pipette gut mischen.</li>
	<li>Autoklav alle chirurgischen Instrumente und den Arbeitsbereich mit 70 % Ethanol zu sterilisieren. Sprühen von chirurgischen Instrumenten und Handschuhe mit 70 % Ethanol häufig oder mit einem Glas Bead Sterilisator bei der Durchführung der folgenden Schritte um sterile Bedingungen zu halten.</li>
</ol>(2) betäubende Tier
<ol><li>Verwenden Sie ein Tischsystem Top Anästhesie, um das Tier mit Isofluran zu betäuben. O2 Tank schalten Sie ein und einstellen Sie der Sauerstoff-Durchflussmesser auf 1 L/min.</li>
	<li>Legen Sie eine Erwachsene weiblichen C57BL/6J Maus in der Induktion-Kammer. Einschalten und Einstellen des Verdampfers Isoflurane auf 3 % für die Induktion. Die Maus sollte in Narkose innerhalb von 2 min. verwalten Buprenorphin Analgetikum (0,1 mg/kg) subkutan nach Entfernung der Maus aus der Induktion Kammer für präemptiven Analgesie. Hinweis: In diesem Protokoll werden weibliche Mäuse verwendet sind einfacher, mit zu arbeiten, während Katheterisierung der Harnröhre. Das Protokoll funktioniert auch für männliche Mäuse, aber besondere Vorsicht ist erforderlich, wenn Schritt 3 ausführen.</li>
	<li>Gelten Sie ophthalmologischen Salbe für die Augen des Tieres um Trockenheit zu verhindern. Platzieren Sie die narkotisierter Maus nach oben auf ein Heizkissen mit seiner Nase in die bugnase. Schalten Sie die Luft Schaltung Isofluran die bugnase durchlaufen zu lassen.</li>
	<li>Der Kopf und die bugnase mit Klebeband zurückhalten. Isofluran Vaporizer auf 2 % für die Wartung einstellen.</li>
	<li>Zurückhalten der Gliedmaßen mit Klebeband. Durchführen Sie Zehe-Prise Tests um sicherzustellen, dass das Tier in Tiefe Narkose und dann rasieren Bauchbereich.</li>
</ol>3. Urin Erschöpfung und Blase spülen
<ol><li>Gelten Sie Gleitmittel für Katheter (24G, Länge von 2,11 cm, Außendurchmesser von 0,045 cm) und sicherzustellen Sie, dass seine äußere Oberfläche vollständig abgedeckt ist.</li>
	<li>Einführen Sie den Katheter in die Harnröhre Öffnung und schieben Sie langsam ein, bis zu welchem Zeitpunkt der Urin durch den Katheter abfließen sollte die Blase erreicht. Drücken Sie vorsichtig den Bauch Urin Erschöpfung helfen.<ol>
<li>Überprüfen Sie, ob der Katheter in die Blase richtig eingesetzt ist, durch die Beobachtung automatischer Urin out fließen. Vermeiden Sie piercing von der Harnröhre und Blase Wand, und wenden Sie Schmiermittel mehrmals, wenn nötig.</li>
	</ol>
</li>
	<li>Verwerfen Sie den Urin mit einer Pipette in ein Abfall Becherglas.</li>
	<li>Pipette 80 µL Phosphat gepufferte Kochsalzlösung (PBS) in das äußere Ende des Katheters, mit dem anderen Ende noch in der Blase. Befestigen Sie eine 1 mL Spritze vorsichtig um den Katheter. Drücken Sie vorsichtig die Spritze, die PBS in der Blase zu injizieren. Lassen Sie einige PBS in den Katheter zu vermeiden, Luftblasen in der Blase.</li>
	<li>Ziehen Sie die Spritze. Warten Sie auf PBS aus der Blase zu entleeren. Drücken Sie vorsichtig den Bauch, PBS zu evakuieren.</li>
	<li>Entsorgen Sie die PBS in den Katheter mit einer Pipette in den Abfall Becher.</li>
	<li>Wiederholen Sie PBS (Waschschritten 3.4-3.6) für zwei weitere Male. Hinweis: Es ist wichtig, während diese Waschschritte sanft arbeiten. Blut in den Katheter oder das Fehlschlagen der automatischen Out-Flüssigkeit fließen in der Regel zeigt die Harnröhre durchbohrt. Es ist auch wichtig zur Vermeidung von Luftblasen, die Einführung, wie sie die Elektroporation Effizienz sinkt.</li>
</ol>(4) Plasmid Lieferung
<ol><li>Gelten Sie 70 % igem Ethanol für die Maus-Bauch und verwenden Sie ein Skalpell, um einen vertikalen Schnitt von 1 cm, öffnen Sie die Bauchhaut oberhalb der Blase machen.</li>
	<li>Pipette mindestens 20 µL Plasmid Plus Trypan blau-Lösung in das äußere Ende des Katheters, und befestigen Sie die Spritze.</li>
	<li>Die Plasmid-Lösung in die Blase zu injizieren. Wenn die blase blau leuchtet, ist die Injektion erfolgreich. Lassen Sie etwas Flüssigkeit in den Katheter zu vermeiden, Luftblasen in der Blase.</li>
	<li>Greifen Sie der äußeren Harnröhrenmündung mit einer Pinzette zu, und entfernen Sie den Katheter und Spritze. Verwenden Sie eine Zeichenfolge an der äußeren Harnröhrenmündung festziehen. Zwei Schleifen mit der Zeichenfolge zu machen und dann mit zwei Knoten binden. Hinweis: Verschärfung der die Harnröhre ist wichtig zur Verhinderung von Rückfluss von Plasmid Flüssigkeit.</li>
	<li>Schalten Sie die Elektroporation-Generator mit den folgenden Parametern: 33V, 50 ms, Arbeitszeit und 950 ms Intervallzeit.</li>
	<li>Halten Sie die Blase mit einer Pinzette und Klemmen Sie die Blase mit zwei Elektroden. Drücken Sie das Fußpedal die Elektroporation durchführen. Hinweis: Die elektrischen Impulse sollen 5 Mal mit kurzen Abständen auftreten, nachdem jedes Pedal drücken. Die Maus kann in diesem Prozess zucken. Schieben das Fußpedal mehr als einmal die Transporteffizienz erhöhen kann, aber zu viele elektrische Impulse können die Gewebe Integrität das Urothel beschädigen.<ol>
<li>Vermeiden Sie jeglichen Kontakt zwischen die Pinzette und die Elektroden, da dies einen Funken zu erzeugen.</li>
	</ol>
</li>
</ol>5. Wiederherstellung
<ol><li>Naht der Bauch- und Haut Eröffnung mit sterilen chirurgischen Naht und Clips. Jod auf die Wunde auftragen</li>
	<li>Entfernen Sie das Tier aus dem Isofluran-System. Verwalten von Buprenorphin Analgetikum (0,1 mg/kg) subkutan, um postoperative Schmerzen zu lindern.</li>
	<li>Halten Sie das Tier auf das Heizkissen und wieder nach Hause Käfig nach vollständiger Genesung. Überprüfen Sie das Tier mindestens einmal täglich für normale Beweglichkeit, trinken und Fütterung Verhalten und keine Anzeichen einer Infektion, bis der Schnitt verheilt ist und entfernen Sie dann die Clips. Verabreichen Sie nachfolgende Injektionen von Buprenorphin Schmerzmittel, wenn das Tier Schmerzen Symptom zeigt, wie putzen, gesteigerte Aggressivität und Vokalisation reduziert.</li>
</ol>

<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. 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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>

Die Autoren erklären keine konkurrierenden Interesse.

Elektroporation verbessert die Durchlässigkeit der Zellmembran und ist eine leistungsstarke Technologie für gen-Electrotransfer-16. Gen-Lieferung in lebenden Nagetiere durch Elektroporation wurde häufig in der Neurobiologie Felder17,18,19,20,21verwendet. Seine Anwendungsmöglichkeiten in vielen anderen Organen haben nicht ausgiebig e...

Gentechnisch veränderte Mausmodelle (GEMMs) spielen eine wesentliche Rolle bei der Erweiterung unseres Wissens über TIERENTWICKLUNG, gen Funktionen in Vivound Mechanismen der Krankheit Fortschreiten1,2. Keimbahn GEMMs werden traditionell in zweierlei Hinsicht entwickelt. Die erste ist die Schaffung von transgenen Mäusen durch man Injektion von DNA Plasmid, gefolgt von der Transplantation von Zygoten in pseudopregnant Weibchen. Die andere ist die Generierung von Knock-Out/Knock-in Mäusen durch homologe Rekombination in embryonalen Stammzellen und die Entwicklung der Chimären. Bedingte ko von Genen in bestimmten Geweben oder Zellen Art von Interesse gehört die Zucht von gentechnisch Allele mit Cre und Flox Standorten über mehrere Generationen. Der gesamte Prozess kann teuer und zeitaufwändig sein, besonders wenn das Ziel ist es, mehrere Gene Gewebe-gezielt. Vor kurzem haben mehrere Organe der Mäuse induzieren Gewebe-spezifische genetische Veränderungen für autochthone Krebs Modellierung3gen Bearbeitung Technologien wie gruppierte regelmäßig dazwischen kurze palindromische Wiederholungen (CRISPR) zugewiesen wurde, 4,5,6,7 oder richtige Krankheit Mutationen in Situ8,9. In diesen Studien wurde die Lieferung der gRNA Vektoren in der Regel durch adenoviralen oder Lentivirale Infektion der Orgel oder hydrodynamischen Tail-Vein Injektion erreicht. Die relative Leichtigkeit der Lieferung der CRISPR-Komponenten auf die Lunge und Leber hat sich verbessert den Komfort und die Effizienz von Krebs in diesen Organen im Vergleich zu den traditionellen Cre-Lox-basierte Mausmodellen Modellierung.
Die Blase Urothel ist wo die meisten Blase Tumoren entstehen. Die Lieferung von DNA-Vektoren an der Blase Urothel für die Krebstherapie Modellierung oder gen scheitert an ein Glykosaminoglykan-Schicht auf der apikalen important Oberfläche fungiert als ein Hindernis für die Einhaltung von infektiösen Viren10,11. Mehrerer Vorbehandlung Agenten wie Syn3 und Dodecyl-β-d-maltosid wurden zur stören diese Barriere und Adenovirus-Infektion Effizienz12,13,14zu erhöhen. Ob Adeno-assoziierten Viren (Flugabwehrpanzer) effektiv die Blase infizieren können, wurde nicht berichtet. Alles in allem wäre eine nicht-viralen basierend Übermittlungsmethode wünschenswert. Hier beschreiben wir eine neuartige Methode, die im Labor für effiziente DNA Plasmid-Lieferung an die Maus Urothel durch Katheterisierung der Harnröhre und Elektroporation entwickelt. Da unser Ansatz nicht chemische Vorbehandlung der Glykosaminoglykan-Schicht oder Virus Produktion involviert ist, es wesentlich vereinfacht die Bereitstellung von DNA Plasmide in der Maus Blase Urothel, und sollte erleichtern verschiedene in-Vivo -Studien Erkrankungen der Blase.

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

Gentechnisch veränderte Mausmodelle (GEMMs) sind sehr wertvoll bei der Enthüllung neuartige biologische Einblicke in die Initiierung und Progression Mechanismen der menschlichen Krankheiten wie Krebs. Transgenen und bedingte Knockout-Mäusen werden häufig seit Überexpression des Gens oder Ablation in bestimmten Geweben oder Zellen in Vivo-Typen. Generierung von Keimbahn Mausmodellen kann Zeit- und kostenaufwändige jedoch. Neue Zuführungen in gen Bearbeiten von Technologien und die Machbarkeit liefern DNA Plasmide durch Virusinfektion haben schnelle Generierung von nicht Keimbahn autochthonen Mausmodellen Krebs für mehrere Organe aktiviert. Die Blase ist ein Organ, das für virale Vektoren aufgrund des Vorhandenseins einer Glykosaminoglykan Schicht deckt das Urothel Zugang zu schwierig gewesen sei. Hier beschreiben wir eine neuartige Methode, entwickelt im Labor für die effiziente Bereitstellung von DNA Plasmide in der Maus Blase Urothel in Vivo. Durch die intravesikale Instillation von pCAG-GFP DNA Plasmid und Elektroporation chirurgisch freigelegten Blase zeigen wir, dass das Plasmid DNA spezifisch in die Blase important Zellen für transiente Expression geliefert werden kann. Unsere Methode bietet eine schnelle und bequeme Möglichkeit für Überexpression und Knockdown von Gene in der Maus Blase und zum Aufbau von GEMMs von Blasenkrebs und anderen urologischen Krankheiten angewendet werden kann.

Um den Erfolg der Gen-Lieferung in die Blase Urothel zu demonstrieren, haben wir die pCAG-GFP15 Plasmid für Elektroporation verwendet. 20 µL Plasmid und Trypan blau Lösung wurde durch die Harnröhre injiziert und 5 elektrische Impulse verabreicht wurden. Nach 48 h wir seziert die Maus Blase und Flecken von GFP Fluoreszenz unter dem Dissektion-Fluoreszenz-Mikroskop beobachtet, während eine Negative Kontrolle Blase, die nicht unterziehen Elektroporation zeigte ke...

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

Wir beschreiben eine neue Methode für die Lieferung von Plasmid DNA in die important-Zellen der Maus Blase in Vivo durch Katheterisierung der Harnröhre und Elektroporation. Freuen Sie sich auf eine schnelle und bequeme Möglichkeit zur Erzeugung von autochthonen Mausmodelle der Blase Krankheiten.

Neuartige Methode der Transport der Plasmid-DNA zu Maus Blase Urothel durch Elektroporation

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

novel-method-plasmid-dna-delivery-to-mouse-bladder-urothelium

Research

JoVE Journal

Biology

8.4K Aufrufe.  University of California, Santa Cruz. Wir beschreiben eine neue Methode für die Lieferung von Plasmid DNA in die important-Zellen der Maus Blase in Vivo durch Katheterisierung der Harnröhre und Elektroporation. Freuen Sie sich auf eine schnelle und bequeme Möglichkeit zur Erzeugung von autochthonen Mausmodelle der Blase Krankheiten.

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

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Forschung

Biologie

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation von Plasmid-DNA in E. coli mit dem Hitzeschock-Methode

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

DNA Transfektion von Säuger Skelettmuskulatur mit 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. 

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 inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

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

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

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

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

Optimierte Reinigung von Plasmid-DNA aus prokaryontischen Kulturen

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

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Direkte Lieferung von MIF Morpholinos Into the Zebrafisch Otocyst durch Injektion und Elektroporation Beeinflusst Inner Ear Entwicklung

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

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 blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Eine praktische und neuartige Methode, um genomische DNA aus Blutentnahme Kits Auszug für 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

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Kultur Verfahren zur embryonalen Maus Herz

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

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

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

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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

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 fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction 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 ...