Diese Arbeit wurde zum Teil von der Naturwissenschaftlichen Stiftung von China (Zuschussnummern 31471703, A0201300537 und 31671854 an JV und LL) und dem 100 Foreign Talents Plan (Zuschussnummer JSB2014012 an JV) unterstützt.

Verfahren, die Tierfächer betreffen, wurden vom Ethischen Komitee des Experimentellen Tierzentrums der Nanjing Agricultural University im Einklang mit den nationalen Richtlinien für das experimentelle Tierschutz (Ministerium für Wissenschaft und Technologie, PR von China, 2006) mit den Tieren, die in einem SPF untergebracht wurden, genehmigt (Berechtigungs-ID: SYXK-J-2011-0037). 1. Cmah Knock-out Maus Modell <ol><li> Verwenden Sie Wildtyp-C57Bl / 6-Mäuse aus dem Vergleichsmedizinzentrum der Universität von Yangzhou (China). HINWEIS : Cmah- Knock-out-Mäuse wurden basierend auf den Informationen aus früheren Cmah- Knock-out-Studien 17 , 19 erstellt und kommerziell unter Verwendung einer CRISPR / Cas9 20- Strategie gewonnen, indem 92 Basenpaare aus Exon 6 des Cmah- Gens entfernt wurden, was ebenfalls der Fall ist Deletiert im menschlichen CMAH- Gen 19 . Positiv F 0 &lt;/ sub&gt; -mice (2 Personen) wurden mit Wildtyp - Mäusen gekreuzt heterozygot F 1 -mice zu erhalten. Nach dem Überqueren von heterozygoten F 1 -Mäuse (5 Individuen) konnte man homozygote Cmah- Knock-out-F 2 -Mäuse erfolgreich erhalten (3 Individuen). </li><li> Führen Sie die Genotypisierung von Mäusen mit genomischer DNA nach dem von Zangala 21 gezeigten Verfahren unter Verwendung eines kommerziellen DNA-Reinigungskits durch. <ol><li> Verstärken Sie ein entsprechendes DNA-Fragment des Cmah- Gens unter Verwendung von kommerzieller DNA-Polymerase und dem Primerpaar 5&#39;gaaagggctcggctctgtataa &#39;&#39; und 5&#39;tttaaatertcccgggtgagaagc3 &#39;. Führen Sie die Genamplifikation unter Verwendung von 34 PCR-Zyklen aus, die aus einer Denaturierung bei 94 ° C für 40 s, einem Glühen bei 63 ° C für 40 s und einer Dehnung bei 72 ° C für 1 min bestehen. </li><li> Visualisierung von PCR-Produkten auf einem 1% Agarosegel. Man sollte ein einzelnes Band (0,5 kb) für das Wildtyp-Individuum, ein Doppelband (0,4 und 0,5 kb) für dieHeterozygoten Knock-out-Individuum und ein einziges Band (0,4 kb) für das homozygote Knock-out-Individuum. </li></ol></li></ol> 2. Probenahme <ol><li> Sammeln Sie die Mausmilch mit dem von Willingham et al. 22 </li><li> Sammle das Mauslebergewebe mit dem von Gonçalves et al. Beschriebenen Protokoll . 23 und Proben bei -80 ° C aufbewahren. </li></ol> 3. Isolierung von Sialinsäuren aus Milch <ol><li> 100 ml einer 2 M Essigsäurelösung durch Zugabe von 12 ml Eisessig in 88 ml destilliertem H 2 O zubereiten. </li><li> 50 μl Milch in ein 1,5 mL Zentrifugenröhrchen geben und 1,2 ml der vorbereiteten wässrigen Essigsäurelösung zugeben. </li><li> Inkubieren Sie die Suspension für 4 h bei 80 ° C und zentrifugieren Sie die Probe bei 14.000 xg für 10 min. </li><li> Übertragen Sie die obersten 1.000 μl des Überstandes in ein frisches 1,5 mL Zentrifugenröhrchen und remDas Lösungsmittel durch Zentrifugalverdampfung bei Raumtemperatur abkühlen lassen, um die Trockenheit zu beenden (abhängig von der angeschlossenen Vakuumpumpe dauert dies 4 - 20 h). Die Probe in 500 μl destilliertem H 2 O auflösen. </li><li> Bereiten Sie eine Mikroanionenaustauschersäule vor. Dieser Schritt wird spezifisch anionische Verbindungen anreichern und kationische und neutrale Verbindungen aus den Milch- und Leberproben entfernen und damit die Selektivität des fluorogenen OPD-Markierungsmittels für die Sialinsäurederivatisierung erhöhen. <ol><li> Übertragen Sie für jede Probe 200 mg des Anionenaustauscherharzes (Dowex 1X8) in leere 3 mL Säulenröhrchen mit einem angehängten Hahn. </li><li> Man gibt 2 ml der in Schritt 3.1 hergestellten wässrigen Essigsäurelösung zu, um die Chloridionen im Harz mit Acetat-Ionen zu ersetzen. Schließen Sie den Hahn, sobald das Lösungsmittel die Oberseite des Harzes erreicht. </li><li> Fügen Sie 2 ml destilliertes H 2 O vor, öffnen Sie den Hahn und stoppen Sie den Fluss, sobald der Großteil des Lösungsmittels erreicht istDie Oberseite des Harzes. Stellen Sie sicher, dass das Harz immer mit Lösungsmittel bedeckt ist. </li><li> Die Harzsäule zwei weitere Male mit 2 ml destilliertem H 2 O zum Entfernen des überschüssigen Acetats waschen. </li></ol></li><li> Übertragen Sie die resultierenden 500 μl Probenlöser aus Schritt 3.4. Auf die Harzsäule auftragen und den Durchfluss verwerfen. Das Harz mit 2 ml destilliertem H 2 O vorsichtig waschen. Die Probenanionen aus dem Harz unter Verwendung von 1 ml einer 50 mM Ammoniumacetatlösung (386 mg Ammoniumacetat, gelöst in 100 ml destilliertem H 2 O) in ein 1,5 ml eluieren Zentrifugenröhrchen. Entfernen Sie das Lösungsmittel durch Zentrifugalverdampfung bei Raumtemperatur bis zur Trockenheit. HINWEIS: Trockene Proben können bei 4-6 ° C für bis zu 12 Monate gelagert werden. </li></ol> 4. Isolierung von Sialinsäuren aus Lebergewebe <ol><li> Vorsicht 20 - 50 mg Mausleber auftauen und in eine Dounce - Gewebemühle (1 ml oder 2 ml Volumen) überführen. Zugabe von 1,2 ml der wässrigen EssigsäurelösungHergestellt in Schritt 3.1 und homogenisieren das Gewebe durch sanfte Scherung für 10 s. Die erhaltene Suspension in ein 1,5 mL Zentrifugenröhrchen überführen. </li><li> Folgen Sie den Schritten 3.3. Auf 3,6. HINWEIS: Trockene Proben können bei 4-6 ° C für bis zu 12 Monate gelagert werden. </li></ol> 5. Vorbereitung eines gemischten Sialinsäure-Standards <ol><li> Wiegen zwischen 5 - 8 mg N-Acetylneuraminsäure auf einem analytischen Gleichgewicht in ein 1,5 mL Zentrifugenröhrchen. Man beachte das genaue Gewicht und füge 164 μl destilliertes H 2 O für jede mg hinzu ( dh wenn das Gewicht von Neu5Ac 7,2 mg beträgt, dann 7,2 x 164 = 1, 181 μl destilliertes H 2 O) zugeben. Daraus ergibt sich eine 20 mM Neu5Ac-Stammlösung, die bei -20 ° C für bis zu 12 Monate gelagert werden kann. </li><li> N-Glykolylneuraminsäure in kleineren Mengen zu einem moderaten Preis erhalten, wie in 1 mg Aliquoten. Füge 154 μl destilliertes H 2 O direkt zu der Verbindung hinzu, um eine ~ 20 mM Neu5Gc-Stammlösung zu erhalten. Übertragen Sie dieLösung in ein 1,5 mL Zentrifugenröhrchen gegeben. Diese Lösung kann bei -20 ° C für bis zu 12 Monate gelagert werden. </li><li> Kombinieren Sie 5 μl aus jeder Stammlösung aus Schritt 5.1 und 5.2 in ein frisches 1,5 mL Zentrifugenröhrchen und entfernen Sie das Lösungsmittel durch Zentrifugalverdampfung bei Raumtemperatur bis zur Trockene. HINWEIS: Der gemischte Sialinsäurestandard kann bis zu 12 Monate bei 4 - 6 ° C gelagert werden. </li></ol> 6. Fluoreszenzderivatisierung von Sialinsäuren <ol><li> Vorbereiten von 10 ml OPD-Lösung, bestehend aus 100 mg o-Phenylendiamin und 208 mg Natriumhydrogensulfit in 10 ml destilliertem H 2 O. </li><li> Füge 20 μl OPD-Lösung zu den aus Mäusemilch gewonnenen Sialinsäureproben (Schritt 3), Mausleber (Schritt 4) oder dem gemischten Sialinsäurestandard (Schritt 5) hinzu. Vortex kräftig für 30 s und inkubieren Proben für 4 h bei 80 ° C im Dunkeln ( dh wickeln die Mikroröhrchen in Aluminiumfolie). </li><li> Lassen Sie die Proben 5 min abkühlen, fügen Sie 80 μ hinzu; L destilliertem H 2 O und die Röhrchen bei 14000 × g für 1 min zentrifugiert. </li><li> Übertragen Sie 80 μl aus dem Überstand in eine 300 μl hochauflösende HPLC-Durchstechflasche. Die derivatisierten Sialinsäureproben können bis zu einer Woche bei 4 - 6 ° C gelagert werden. </li></ol> 7. HPLC-Analyse von Sialinsäurederivaten <ol><li> Analysieren Sie die Proben mit einem Standard-HPLC-System, das mit einem Online-Fluoreszenzdetektor verbunden ist. </li><li> Verwenden Sie für die Analyse eine Umkehrphasen-C18-Säule mit den Standardabmessungen von 250 mm Länge und 4,6 mm Durchmesser. </li><li> Vorbereitung des Lösungsmittels A durch Verdünnen von 200 ml Stammlösung mit 800 ml LCMS-Wasser (LCMS - Flüssigchromatographie-Massenspektrometrie). Die Stammlösung selbst kann wie folgt hergestellt werden: <ol><li> Füge 46 g Ameisensäure zu 800 ml LCMS-Klasse H 2 O hinzu. </li><li> Stellen Sie den pH-Wert auf 4,5 durch tropfenweise Zugabe von Ammoniumhydroxidlösung (pur. Pa) ein. </li><li> Übertragen Sie das Lösungsmittel auf einMesszylinder und füllen bis zu 1.000 ml mit LCMS-Klasse H 2 O. Diese Stammlösung kann bei 4 - 6 ° C für bis zu 3 Monate gelagert werden. </li></ol></li><li> Für Lösungsmittel B, verwenden Sie LCMS-Grad Acetonitril. </li><li> Trennen Sie die Sialinsäurederivate bei einer 1 mL / min Fließgeschwindigkeit mit folgender Gradientenelution: <ol><li> Beginnen Sie mit der Zugabe von 10% des Lösungsmittels B, das dem Lösungsmittel A gemischt wurde. </li><li> Von 0 min bis 15 min erhöht man allmählich den Anteil des Lösungsmittels B mit einem linearen Gradienten auf 60%. </li><li> Von 15 min auf 16 min steigern schnell den Anteil an Lösungsmittel B mit einem linearen Gradienten von 60% bis 90%. Dies initiiert das Waschen der HPLC-Säule. </li><li> Um die HPLC-Säule weiter zu waschen, halten Sie den Füllstand des Lösungsmittels B bei 90% zwischen 16 min und 18 min, bevor Sie den Füllstand des Lösungsmittels B wieder auf 10% zwischen 18 min und 19 min reduzieren. </li><li> Die HPLC-Säule auf die Ausgangsbedingungen von 10% B zwischen 19 und 24 min re-equilibrieren. </li></ol></li><li> Im50 μl Probe in das HPLC-System geben. </li><li> Überwachen Sie die Eluenten mit den Fluoreszenzdetektor-Anregungs- / Emissionswellenlängen von 373/448 nm und die derivatisierten Sialinsäuren können bei den ungefähre Retentionszeiten von 9 min (für Neu5Gc-OPD) und 10 min (für Neu5Ac-OPD) erwartet werden. </li><li> Berechnen Sie die relative Menge von Neu5Gc (F Neu5Gc ) aus den Fluoreszenz-Peakflächen des Neu5Ac (A Neu5Ac ) und Neu5Gc (A Neu5Gc ) wie folgt: F Neu5Gc [%] = 100 × A Neu5Gc / (A Neu5Ac + A Neu5Gc ). Im Falle der Milch- und Leberproben aus der homozygoten Cmah- Knock-out-Maus sollte F Neu5Gc 0% betragen , während die Werte für F Neu5Gc von heterozygoten und Wildtyp-Mäusen je nach Mausalter und Gewebetyp stark variieren können (zwischen 2% Und&gt; 90%) mit erwarteten Fehlerrändern von ± 12%. </li></ol>

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Das hier beschriebene Protokoll erlaubt die phänotypische Beurteilung von homozygoten Cmah- Knock-out-Mäusen durch Analysieren und Quantifizieren der relativen Mengen an Neu5Gc Milch und Leberproben. Die Analyse wurde unter Verwendung eines Standard-HPLC-Setups mit Fluoreszenzdetektion durchgeführt. Der wichtigste Schritt dieses Verfahrens ist die Herstellung der Anionenaustauschsäulen und die Durchführung der Anionenaustauschchromatographie; Um das Harz richtig zu regeln und die richtigen Wasch- und Eluti...

N-Acetylneuraminsäure (Neu5Ac) und N-Glykolylneuraminsäure (Neu5Gc) sind die häufigsten Sialinsäuren bei den meisten Säugetieren 1 . Obwohl es in der Lage ist, Neu5Ac endogen zu synthetisieren, sind die Menschen nicht in der Lage, Neu5Gc aufgrund einer primären Exon-Deletion auf dem CMAH- Gen, das für eine CMP-Neu5Ac-Hydroxylase 2 , 3 kodiert , zu produzieren . Allerdings können tierische Lebensmittelprodukte diätetische Quellen von Neu5Gc 4 , 5 , 6 sein , was zur Herstellung von Anti-Neu5Gc-Antikörpern führt und daher eine Immunantwort gegen Neu5Gc 7 auslöst. Diese diätetische Wirkung von Neu5Gc wird vermutlich zur chronischen Entzündung und verschiedenen anderen Krankheiten beitragen 8 , 9 , 10 . Um die Wirkungen von Neu5Gc in h zu verstehenUmans, ein Tiermodell für die systematische Untersuchung der Wirkungen von lebensmittelhaltigen Sialinsäuren ist höchst wünschenswert. Obwohl die auf der Polymerase-Kettenreaktion (PCR) basierenden Protokolle zur Analyse von Knock-out-Mäusen gut etabliert sind und ein bequemer Weg für die genotypische Beurteilung ist, erfordert die funktionelle Analyse des Phänotyps auf dem metabolischen Niveau spezifischere Analysemethoden. Der Phänotyp eines Cmah- Knock-out-Mausmodells kann durch Isolierung und Analyse der Zusammensetzung von Sialinsäuren in Leber- oder Milchproben beurteilt werden. Mehrere Verfahren zum Nachweis von Sialinsäuren in tierischen Geweben wurden bisher beschrieben: Die Umsetzung von Sialinsäuren mit Resorcin 11 oder Thiobarbitursäure 12 führt zur Bildung eines chromophoren Produktes und kann einfach unter Verwendung eines plateraderbasierten Aufbaus analysiert werden, aber nur das gesamte Sialis Säuregehalt kann bestimmt werden. Alternativ wurde auch die Analyse von Sialinsäuren mittels Gaschromatographie beschrieben13 , MALDI-ToF-Massenspektrometrie 14 oder amperometrische Methoden 15 . Die am häufigsten angewandten Sialinsäureanalyseverfahren basieren jedoch auf der hydrolytischen Freisetzung, gefolgt von der Fluoreszenzderivatisierung und der nachfolgenden Hochleistungsflüssigkeitschromatographie 16 , 17 , 18 .

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:147px;">Chemicals:</td>
			<td style="width:144px;"></td>
			<td style="width:143px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-acetylneuraminic acid</td>
			<td>Sigma</td>
			<td>A0812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-glycolylneuraminic acid</td>
			<td>Sigma</td>
			<td align="right">50644</td>
			<td>1 mg aliquot should be sufficient</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">o-Phenylenediamine</td>
			<td>Sigma</td>
			<td align="right">694975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydrogen sulfite</td>
			<td>J&#38;K Scientific Ltd</td>
			<td align="right">75234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3 mL SPE tubes</td>
			<td>Supelco</td>
			<td>Sigma 57024</td>
			<td>empty solid phase extraction columns</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer stopcock</td>
			<td>Sigma</td>
			<td>S7396</td>
			<td>to stop the flow of the SPE tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dowex 1X8</td>
			<td>Dow Chemicals</td>
			<td>Sigma 44340</td>
			<td>200-400 mesh</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dounce tissue grinder</td>
			<td>Sigma</td>
			<td>D8938</td>
			<td>tight fit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>#5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Phenomenex</td>
			<td>Hyperclone ODS</td>
			<td>250x4.6 mm</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LCMS-grade H2O</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td>#100029</td>
			<td>Hypergrade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium hydroxide solution</td>
			<td>Fluka</td>
			<td>#44273</td>
			<td>puriss. P.a.</td>
		</tr>
	</tbody>
</table>

determination of sialic acids in liver and milk samples of wild-type and cmah knock-out mice.

CMAH (Cytidinmonophosphat-N-acetylneuraminsäure-hydroxylase) ist verantwortlich für die Oxidation von Cytidinmonophosphat-N-acetylneuraminsäuren bei Säugetieren. Jedoch kann der Mensch Cytidinmonophosphat-N-acetylneuraminsäure nicht zu Cytidinmonophosphat-N-glykolylneuraminsäure aufgrund einer primären Exon-Deletion des CMAH- Gens oxidieren. Um die Effekte und Implikationen des Mangels an CMAH-Aktivität genauer zu verstehen, ist ein Cmah- Knock-out-Modell bei Mäusen von großem Interesse an Grund- und angewandter Forschung. Das Analyseverfahren zur Bestimmung des Phänotyps dieses Mausmodells wird hier im Detail beschrieben und basiert auf dem Nachweis sowohl der N-Acetylneuraminsäure als auch der N-Glykolylenuraminsäure in der Leber und der Milch von Wildtyp- und Cmah- Knock-out-Mäusen. Endogene Sialinsäuren werden freigesetzt und mit o-Phenylendiamin derivatisiert, um fluorogene Derivate zu erzeugen, die anschließend mittels HPLC analysiert werden können. Das vorgestellte Protokoll kann seinAuch für die Analyse von Milch und Gewebeproben aus verschiedenen anderen Ursprüngen angewendet und kann von Nutzen sein, um die ernährungs- und gesundheitlichen Auswirkungen von N-Glykolylneuraminsäure zu untersuchen.

Ein schematischer Überblick über das beschriebene Analyseverfahren ist in Abbildung 1 dargestellt und beinhaltet die Isolierung von Sialinsäuren aus Milch- und Leberproben von Wildtyp- und Cmah- Knockout-Mutanten-Mäusen sowie die Fluoreszenz-Derivatisierung und HPLC-Analyse dieser Komponenten. Abbildung 2 und Abbildung 3 zeigen repräsentative HPLC-Chromatogramme von derivatisierten Sia...

Watch this Scientific Journal Video about Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. at JoVE.com

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

Wir beschreiben ein HPLC-basiertes Verfahren zur Bestimmung von N-Acetylneuraminsäure und N-Glykolylenuraminsäure in Mausleber und Milch.

Bestimmung von Sialinsäuren in Leber- und Milchproben von Wildtyp und<em&gt; CMAH</em&gt; Knock-out Mäuse.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

determination-sialic-acids-liver-milk-samples-wild-type-cmah-knock

Research

JoVE Journal

Biology

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

9.7K Aufrufe.  Nanjing Agricultural University.  Wir beschreiben ein HPLC-basiertes Verfahren zur Bestimmung von N-Acetylneuraminsäure und N-Glykolylenuraminsäure in Mausleber und Milch. 

Serie: Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

Die Quantifizierung der γH2AX Foci in Gewebeproben

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Forschung

Biologie

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Ermittlung der Auswirkungen von BRCA1-Mutationen auf homologe Rekombination unter Verwendung von Zellen, die endogenes Wildtyp-BRCA1 Express

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Konzentrationsbestimmung von Nukleinsäuren und Proteinen Mit dem Micro-Volumen Bio-spec Nano-Spektralphotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

In vivo Leber Endozytose durch Reinigung von Leberzellen durch Leberperfusion Gefolgt

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Ein PCR-basierte Genotypisierung Methode, zwischen Wildtyp-und Ziersorten Unterscheiden von Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Einrichtung zur Rückgewinnung von kryokonservierten Proben versehentlich fallengelassen in Kryo-Lagerbehälter

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Milchauffangmethoden für Mäuse und Reeves Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Bestimmung der Fettsäureoxidation und Lipogenese in Maus primären Hepatozyten

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Milchsammlung in der Ratte Mit Kapillarröhrchen und Schätzung der Milchfettgehalt von Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtration Isolierung von Nukleinsäuren: Eine einfache und schnelle DNA-Extraktionsmethode

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Bestimmung von Sialinsäuren in Leber- und Milchproben von Wildtyp und

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