Die Autoren danken der Molekularpathologie und Bildgebung Kern der UPenn NIDDK-Centrum für Molekulare Studien in Verdauungs-und Lebererkrankungen (P30 DK50306) für die Unterstützung bei Bildgebung. Diese Arbeit wurde durch Zuschüsse aus dem National Institutes of Health (R01 DK-092111) und von der Fred und Suzanne Biesecker Pediatric Liver Center (zu RGW) und durch eine Gemeinschaft aus der Kindheit Liver Disease Research and Education Network (SK) unterstützt.

Das gesamte Verfahren wird bei Raumtemperatur durchgeführt, sofern nicht anders angegeben. Alle Tier Arbeit sollte unter menschenwürdigen Bedingungen, unter einem Protokoll von der örtlichen Institutional Animal Care und Verwenden Committee (IACUC) zugelassen sind. 1. Herstellung von Ausrüstung und Lösungen <ol><li> Richten Sie die Maus OP-Tisch in der Nähe der Gewebekultur Kapuze und Inkubator (Abbildung 1A). Hinweis: Erforderliche Ausrüstung umfasst ein Binokular, eine Lichtquelle, 12,5 cm lange, gerade Iris Schere, 6 Zoll nicht gezackten gebogenen Pinzette mit feinen Spitzen, und 4-Zoll-gezähnt Zange mit gebogenen Spitzen (Abbildung 1B). </li><li> Platzieren sterilisierten Instrumente in einem Becherglas von 70% Alkohol auf dem Operationstisch. </li><li> In drei sterile 60 mm Petrischalen mit sterilen Isolationsmedien (Tabelle 1); legen Sie zwei am OP-Tisch und eine Kapuze, die alle auf dem Eis. </li><li> Zeigen Rattenschwanz Kollagen,sterile 10 x phosphatgepufferter Salzlösung (PBS), sterilem Wasser, steriler 1 N NaOH und Gallen Epithelzellen (BEC)-Medien (Tabelle 1) in der Gewebekultur Haube. Hinweis: Das Kollagen sollte auf Eis. </li></ol> 2. Gallenwegs Isolierung und Kultur <ol><li> Euthanize neugeborenen Mäusen zwischen 0-3 Tagen des Lebens, durch die Einwirkung von Kohlendioxid bis zum Unbewussten durch Genickbruch, pro IACUC Richtlinien (1C), gefolgt. Hinweis: ältere Mäuse können in Abhängigkeit von bestimmten experimentellen Anforderungen verwendet werden. </li><li> Zeigen Tier in Rückenlage und machen einen kleinen horizontalen Schnitt Überqueren der Mittellinie im Bereich des Beckens. Erweitern Sie dieses Schnitt seitlich und bis beide Seiten des Bauches, bilden eine U-förmige Klappe. Schalten Klappe bis die Bauchorgane aus. Sobald das Abdomen geöffnet ist, vorsichtig bewegen die Eingeweide aus der Bauchhöhle in Richtung der rechten Seite des Tieres zur besseren Visualisierung der Leber und der Gallengang. Hinweis: es kann notwendig sein, um die seitlichen Einschnitte in den Brustkorb zu verlängern, um eine bessere Identifikation der Leber zu erhalten. </li><li> Verwendung der Präpariermikroskop mit den gemeinsamen Gallengang, Leber und Darm. Identifizieren Sie den Gallengang erste (1D), mit Vorsicht, da die Strukturen sind sehr empfindlich. Mit den gezackten und nicht gezackten Zangen, akribisch sauber und holen Sie das Bindegewebe, einschließlich Fett rund um die Galle. Verwenden Sie eine Zange, um den Kanal und die andere zu reinigen halten. </li><li> Verwenden Sie eine ähnliche Technik, um die Gallenblase zu reinigen. </li><li> Legen Sie die gereinigten Produkte und Gallenblase in den ersten Teller der Isolationsmedien auf Eis. Massieren Sie die Strukturen mit den nicht gezahnten Pinzette während in den Medien, um weiter zu entfernen Bindegewebe und Galle aus der Gallenblase zu entfernen. </li><li> Übertragen Kanäle und Gallenblase in die zweite Schale der Isolation Medien. </li><li> Nach der Isolierung von Leitungen und Gallenblasen aus 5 Tieren, übertragen Sie dieStücke mit dem dritten Petrischale, in der Haube. Hinweis: Wenn Zellen von mehr als 5 Tieren isoliert werden sollen, tun in den Reihen, die mit frischen Lösungen für jede Gruppe von 5 Tieren. </li><li> Bereiten dicken Kollagengelen in der Gewebekultur Haube: Hinweis: jeder Größe Gericht verwendet werden können. Eine 60-mm-Kulturschale mit einem Endvolumen von 3 ml Kollagen für jede Gruppe von 5 Neugeborenen funktioniert gut. Dies sollte frisch zubereitet werden, zum Zeitpunkt der Einbettung frisch isolierten Leitungen. <ol><li> Platzieren Rattenschwanzkollagen, sterile 10 x phosphatgepufferter Kochsalzlösung (10x PBS), steril dH 2 O und 1 N NaOH sterile auf Eis. </li><li> Berechnung der Volumina von dH 2 0, 10x PBS, NaOH und Kollagen für Gele mit einer Endkonzentration von 2 mg / ml Kollagen in 1 × PBS erforderlich, mit einem Volumen von 1 N NaOH gleich 0,023 mal dem Volumen des Kollagens zugesetzt. Hinweis: Wenn alternative Quelle von Kollagen verwendet wird, befolgen Sie die Anweisungen des Herstellers für die Neutralisation und Gel-Bildung. </li><li> Mix zusammen dH 2 0, 10x PBS und 1 N NaOH, dann fügen Kollagen und Pipette auch, um sicherzustellen, sogar Vermischung. </li></ol></li><li> Sobald Kollagenlösung hat zu einer gelartigen Konsistenz bei Raumtemperatur verdickt, die sofort einbetten Kanäle im Kollagen. </li><li> Legen in den Inkubator bei 37 ° C, 5% CO 2 für 30 min, um das Kollagen zu ermöglichen sich zu verfestigen. </li><li> Sobald Gel (Farbwechsel von klar bis weiß), Überlagerung mit 3,5 ml BEC Medien (Tabelle 1) verfestigt und bei 37 ° C, 5% CO 2. </li><li> BEC Medien entfernen 3 mal pro Woche und ersetzen mit 3,5 ml frischem Medium. </li></ol> 3. Isolaprimär Cholangiozyten von Dick Kollagengele Hinweis: innerhalb von 3 Wochen, werden Blätter Cholangiozyten (Zellen mit großen Kernen, die sich direkt von Embedded-Kanäle) in der dicken Kollagen sichtbar sein. Wenn es eine große Zahl von Fibroblasten in die Schüssel, sollte sie verworfen werden. Wenn ein paar Regionen der Schüssel habenFibroblasten-Wachstums, können diese mit einem Skalpell geschnitten und vor der Aufspaltung der Cholangiozyten entfernt werden. <ol><li> Bereiten dünnen Kollagengelen: <ol><li> Verdünnen Kollagen in einer Konzentration von 1 mg / ml mit steriler PBS. Hinweis: Verwenden Sie 1 ml für 100-mm-Platte; entsprechend für andere Gerichte Größe anpassen. </li><li> Verbreiten Sie die Kollagenlösung gleichmäßig mit einer Zelle Treuer (große Geschirr) oder Pipettenspitze (kleine Gerichte), dann lassen bei Raumtemperatur für 10 min. </li><li> Platte mit DMEM/F12 und bei 37 ° C, 5% CO 2 für 10 min. </li><li> Entfernen Platte von Inkubator und waschen mit 1x PBS. Unmittelbar absaugen 1x PBS. </li><li> Mantel mit einem zweiten dünnen Schicht aus Kollagen, die Verbreitung durch Drehen der Platte oder Platzierung Platte auf einem Schüttler. Bei 37 ° C, 5% CO 2 für 10 min. </li><li> Nach Kollagen verfestigt hat, Abdeckplatte mit DMEM/F12 und bei 37 ° C, 5% CO 2 für 30 min. </li></ol></li><li> Split-Zellen von thick Kollagengelen: <ol><li> Mit Zellkulturzellschaber oder Spachtel, kratzen Kollagengel vorsichtig von der Platte, so daß sie schwimmt. </li><li> Bereiten Collagenase-Lösung durch Verdünnen Typ XI Kollagenase (siehe Materialliste) auf 10 mg / ml mit DMEM/F12. Gut mischen und Sterilfilter. </li><li> 1 ml der Collagenase-Lösung (oben) pro 60 mm-Schale von Zellen in dicken Kollagen. Die BEC-Medien (3 ml, das sollte sein) nicht entfernen. Inkubieren Schale für 30 min bei 37 ° C, 5% CO 2. </li><li> Entfernen Lösung, die Zellen und legen in 15 ml konischen Röhrchen. </li><li> Spin down bei 2.200 xg für 5 min. Überstand verwerfen und Pellet erneut aussetzen ähnliches Volumen von DMEM/F12. </li><li> Spinnlösung erneut bei 2200 · g für 5 min. Überstand entfernen. </li><li> Pellet in 3 ml 0,5% Trypsin und Inkubation bei 37 ° C, 5% CO 2 für 5 min. Nach der Inkubation Pipette die Lösung auf und ab, brechen die Blätter Cholangiozyten. 5 ml BEC Medien einnd Spinnlösung bei 1.500 × g für 5 min. </li><li> Überstand entfernen und Pellet in DMEM/F12, dann spinnen Lösung bei 1.500 xg für 5 min. </li><li> Überstand entfernen und Pellet erneut aussetzen BEC Medien (Tabelle 1). Teller Zellen auf bisherigen dünnen Kollagen Gele. </li></ol></li></ol> 4. Passage und Lagerung von Zellen Hinweis: bei Bedarf können die Zellen auf dünnen Kollagengelen aufgeteilt werden. <ol><li> Vor der Inkubation in 0,25% Trypsin bei 37 ° C für 10-15 min waschen Platten mit sterilem PBS. </li><li> Neutralisieren mit einem gleichen Volumen von BEC Medien und pelletieren bei 1.500 × g für 5 min. Mit DMEM/F12 spülen Pellet, dann pelletieren bei 1.500 × g für 5 min. </li><li> Die Zellen in BEC Medien für die Verteilung an Kollagen beschichtete Zellkulturschalen. Hinweis: Alternativ können die Zellen in Speichermedien resuspendiert und in einem -80 ° C Gefrierschrank oder flüssiger Stickstoff (Tabelle 1) gespeichert werden. </li></ol>

<ol>
	<li>Paradis, K., Sharp, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+duct-like+structure+formation+after+isolation+of+bile+ductular+cells+from+a+murine+model.">In vitro duct-like structure formation after isolation of bile ductular cells from a murine model.</a> J. Lab. Clin. Med. 113 (6), 689-694 (1989).</li><li>Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+polarized+primary+cultures+of+rat+intrahepatic+bile+duct+epithelial+cells.">Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.</a> Lab. Invest. 74 (1), 303-313 (1996).</li><li>Kumar, U., Jordan, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+biliary+epithelial+cells+from+the+biliary+tract+fraction+of+normal+rats.">Isolation and culture of biliary epithelial cells from the biliary tract fraction of normal rats.</a> Liver. 6 (6), 369-378 (1986).</li><li>Ishii, M., Vromen, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+morphologic+characterization+of+bile+duct+epithelial+cells+from+normal+rat+liver.">Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver.</a> Gastroenterology. 97 (5), 1236-1247 (1989).</li><li>Strazzabosco, M., Fabris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+bile+ducts:+Essentials+for+the+clinical+hepatologist.">Development of the bile ducts: Essentials for the clinical hepatologist.</a> J. Hepatol. 56 (5), 1159-1170 (2012).</li><li>Carpentier, 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=Embryonic+ductal+plate+cells+give+rise+to+cholangiocytes,+periportal+hepatocytes,+and+adult+liver+progenitor+cells.">Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.</a> Gastroenterology. 141 (4), 1432-1438 (2011).</li><li>Cho, W. K., Mennon, A., Boyer, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+polarized+bile+duct+units+from+mouse+liver.">Isolation of functional polarized bile duct units from mouse liver.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 280 (2), (2001).</li><li>Karjoo, S., Hand, N. J., Loarca, L., Russo, P. A., Friedman, J. R., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extra-hepatic+cholangiocyte+cilia+are+abnormal+in+biliary+atresia.">Extra-hepatic cholangiocyte cilia are abnormal in biliary atresia.</a> J. Pediatr. Gastroenterol. Nutr. 57 (1), 96-101 (2013).</li><li>Sutton, M. E., op den Dries, S., Koster, M. H., Lisman, T., Gouw, A. S., Porte, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+human+extrahepatic+biliary+epithelium:+the+peribiliary+glands+as+progenitor+cell+compartment.">Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.</a> Liver Int. 32 (4), 554-559 (2012).</li><li>Cardinale, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stem/progenitor+cells+in+human+biliary+tree+give+rise+to+hepatocytes,+cholangiocytes,+and+pancreatic+islets.">Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.</a> Hepatology. 54 (6), 2159-2172 (2011).</li></ol>

Die Autoren erklären, dass sie keine Interessen haben.

Beschrieben wird hier eine Technik, um reine Cholangiozyten aus den extrahepatischen Gallenwege von Mäusen Mäuse aller Altersgruppen, einschließlich Neugeborenen zu isolieren. Die Technik bietet den Vorteil, dass extrahepatischen Cholangiozyten getrennt von intrahepatischen Cholangiozyten untersucht werden, und können Studien zu wichtigen Unterschiede zwischen diesen Populationen von Zellen identifizieren zu erleichtern. Wir haben vor kurzem eine Studie veröffentlicht, demonstrieren verringert Flimmerhärchen in ex...

Die Entstehung und Entwicklung der intra-und extrahepatischen Cholangiozyten unterscheiden sich deutlich. Die Leber entwickelt sich aus einer Divertikel des ventralen Vorderdarm Endoderm 5. Die Schwanzbereich der Divertikel bildet die extrahepatischen Gallenwege, während der Oberkopf erzeugt die intrahepatischen Gallenwege 5. Intrahepatische Kanal Cholangiozyten werden aus Vorläuferzellen rund um den duktalen Platte in den peri-Portal Bereiche 6 abgeleitet. Diese Zellen haben die Fähigkeit, entweder in Hepatozyten und Cholangiozyten 6 unterscheiden. Dies hat erhebliche klinische Implikationen da cholangiopathies kann gezielt eine Kategorie von Cholangiozyten. Zum Beispiel Gallengangsatresie betrifft zunächst die extrahepatischen Kanäle des Neugeborenen, während Alagille Syndrom betrifft intrahepatischen Gänge. Es gibt mehrere Beschreibungen der Isolierung der intrahepatischen Gallengänge und Cholangiozyten Einheiten von Mäusen und Ratten. Investigators haben einzelne Zellen durch Kanaltrennung und anschließender Auswuchs isoliert oder durch Leber und Verdauung Antikörper nach unten ziehen von Cholangiozyten Expression spezifischer Zelloberflächenmarker 4.1. Funktionelle und polarisiert Gallengang-Einheiten wurden von Leber Verdauung und Größe 7 Filtration isoliert. Gallengang-Einheiten in der Lage sind zu reagieren auf Reize sekretorischen und zeigen Fluidsekretion 7, während isoliert Cholangiozyten wurde gezeigt, ductularen Strukturen in vitro 1,2 zu entwickeln. Einschränkungen dieser Methoden sind die Notwendigkeit für spezialisierte technische Expertise und spezielle Ausrüstung für Leberperfusion mit Verdauungsenzymen. Darüber hinaus gibt es ein Risiko der Kontamination mit mesenchymalen Zellen 3. Verfahren extrahepatische Gallengang Cholangiozyten zu isolieren, und zwar von Neugeborenen extrahepatischen Gallenwege, ist bisher nicht beschrieben worden. Dieses Papier skizziert eine vereinfachte Methode für die Neugeborenen eine isolierens sowie erwachsene extrahepatischen Gallengangzellen bei hoher Reinheit. Diese Technik wird die Untersuchung der Unterschiede zwischen den intra-und extrahepatischen Cholangiozyten und Forschung in Mechanismen von Krankheiten wie biliäre Atresie, die extrahepatischen Kanäle beinhalten erleichtern.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="942">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Name of Material/Equipment</td>
			<td style="width:323px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM/F12 (1:1)</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11320-033</td>
			<td>500ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">FBS</td>
			<td style="width:323px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>25 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM with non-essential amino acids</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11140-019</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Insulin-transferrin-selenium</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">51300-044</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Na Pyruvate</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-000-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Chemically-defined lipid concentrate</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11905-031</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin-Streptomycin</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">30-002-CI</td>
			<td>5ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gentamicin</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>0.2ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanolamine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">E9508-100ml</td>
			<td>0.13ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM vitamin solution</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11120-052</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Soybean trypsin inhibitor</td>
			<td style="width:323px;">Biowhittaker</td>
			<td style="width:119px;">17-605E</td>
			<td>5ml, used in BEC media. Solvent is PBS, mix to 5mg/ml stock concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">L-glutamine</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-005-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bovine pituitary extract</td>
			<td style="width:323px;">Gemini</td>
			<td style="width:119px;">500-102</td>
			<td>1.1ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dexamethasone</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">D4902</td>
			<td>0.5ml, used in BEC media, Stock conc 393ug/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">3 3&#39;,5-triiodo-L-thyronine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">T6397</td>
			<td>0.5ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Epidermal growth factor</td>
			<td style="width:323px;">millipore</td>
			<td style="width:119px;">01-101</td>
			<td>0.5ml, used in BEC media, 25ug/ml dilute with DMEM F12+1%BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forskolin</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">F6886</td>
			<td>5ml, used in BEC media, use at stock concentration of 0.411mg/ml and dilute with DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fungizone</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15290-018</td>
			<td>1ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rat-tail collagen</td>
			<td style="width:323px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354236</td>
			<td>variable depending on concentration of collagen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">PBS 10X</td>
			<td style="width:323px;">USB Corporation</td>
			<td align="right" style="width:119px;">75889</td>
			<td>use at 10x, sterlie, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">dH2O</td>
			<td style="width:323px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">NaOH 10N</td>
			<td style="width:323px;">Fischer Scientific</td>
			<td style="width:119px;">ss255-1</td>
			<td>Dilute to 1N, sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">collagenase type XI from Clostridium histolyticum</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td>dilute in DMEM and sterile filter before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (1x) 0.25%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">25200-056</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (10x) 0.5%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15400-054</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;"></td>
			<td style="width:323px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dissecting microscope</td>
			<td style="width:323px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Light source (fiberoptic illuminator)</td>
			<td style="width:323px;">Schott-Fostec</td>
			<td style="width:119px;">Ace EKE LR 92240</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">12.5 cm straight iris scissors</td>
			<td style="width:323px;">Kent Scientific</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">6&#34; non-serrated curved forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">4&#34; serrated stainless forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
	</tbody>
</table>

isolation of neonatal extrahepatic cholangiocytes

Die intra-und extrahepatischen Gallengänge in der Leber sind entwicklungspolitisch deutlich und differentiell durch bestimmte Krankheiten betroffen sein. Jedoch Unterschiede zwischen intra-und extrahepatischen Cholangiozyten und zwischen Neugeborenen und Erwachsenen-Zellen, sind nicht gut verstanden. Methoden zur Isolierung von Cholangiozyten von intrahepatischen Gallengänge sind gut etabliert 1-4. Isolierung von extrahepatischen Gangzellen, insbesondere aus der Neugeborenen ist noch nicht beschrieben worden, obwohl dies von großem Nutzen für das Verständnis der Unterschiede zwischen verschiedenen Bevölkerungsgruppen und Cholangiozyten bei der Untersuchung von Krankheiten wie Gallengangsatresie, die die extrahepatischen Zielkanäle erscheinen. Beschrieben wird hier eine optimierte Technik, um sowohl Neugeborenen und Erwachsenen Maus extrahepatischen Gallengangzellen zu isolieren. Diese Technik ergibt eine reine Zellpopulation mit minimaler Verunreinigung aus mesenchymalen Zellen wie Fibroblasten. Diese method ist auf die Beseitigung der extrahepatischen Gallengänge und, gefolgt von sorgfältige Dissektion und Schaben, Fett und Fibroblastenschichten zu entfernen basiert. Strukturen werden in dicken Schichten von Kollagen und kultiviert für ca. 3 Wochen eingebettet Auswuchs Cholangiozyten in Monoschichten, die dann mit Trypsin behandelt werden kann und wieder überzogen für den experimentellen Einsatz zu ermöglichen.

Die Verwendung dieses Protokoll Ergebnisse bei der Isolierung von einer Bevölkerung von neonatalen Maus extrahepatischen Cholangiozyten mit ausgezeichneter Reinheit, wie von K19 Immunfluoreszenzfärbung nachgewiesen (2A und 2B); wir ähnliche Ergebnisse erzielen Isolierung von Zellen aus erwachsenen Mäusen. Wir haben beobachtet, dass es dauert 3 Wochen für Zellen aus frisch isolierten Gallengänge zu Monoschichten auf dicken Kollagen-Gele bilden. Die Cholangiozyten wachsen in einer linearen Weise in ...

Watch this Scientific Journal Video about Isolation of Neonatal Extrahepatic Cholangiocytes at JoVE.com

Isolation of Neonatal Extrahepatic Cholangiocytes

Eine Technik, um Cholangiozyten aus den extrahepatischen Gallengänge von neugeborenen Mäusen zu isolieren, wird beschrieben. Die Kanäle sind sorgfältig seziert und dann Zellen werden durch Auswuchs in dicken Kollagen-Gelen isoliert. Diese Methode bietet ein nützliches Werkzeug für das Studium extrahepatischen Gallengang Entwicklung und Pathologie.

Isolierung von Neugeborenen Extrahepatische Cholangiozyten

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

isolation-of-neonatal-extrahepatic-cholangiocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.2K Aufrufe.  The Children's Hospital of Philadelphia.  Eine Technik, um Cholangiozyten aus den extrahepatischen Gallengänge von neugeborenen Mäusen zu isolieren, wird beschrieben. Die Kanäle sind sorgfältig seziert und dann Zellen werden durch Auswuchs in dicken Kollagen-Gelen isoliert. Diese Methode bietet ein nützliches Werkzeug für das Studium extrahepatischen Gallengang Entwicklung und Pathologie. 

Serie: Isolation of Neonatal Extrahepatic Cholangiocytes

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Neutrophile Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Forschung

Biologie

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Isolierung von Valvular Endothelzellen

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolierung und Kultivierung von pulmonaler Endothelzellen aus neugeborenen Mäusen

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolierung von Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Isolierung und biophysikalische Untersuchung der Cuticula

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolation und Kultur der Neugeborenen-Maus-Kardiomyozyten

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolierung von embryonalen ventrikuläre Endothelzellen

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolation der Lungenarterie glatten Muskelzellen aus neugeborenen Mäusen

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolierung von murinen Ventil Endothelzellen

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.