Name: determination of sialic acids in liver and milk samples of wild-type and cmah knock-out mice.
Uploaded: 2017-07-14T15:00:00
Description: 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

Dit werk werd gedeeltelijk ondersteund door de Natuurwetenschappelijke Stichting van China (subsidie ​​nummers 31471703, A0201300537 en 31671854 voor JV en LL) en het 100 Foreign Talents Plan (subsidienummer JSB2014012 tot JV).

Procedures waarbij dieren betrokken zijn, zijn goedgekeurd door het Ethisch Comité van het Experimentele Dierscentrum van de Landbouwuniversiteit van Nanjing overeenkomstig de nationale richtlijnen voor experimentele dierenwelzijn (Ministerie van Wetenschap en Technologie, PR van China, 2006) bij de dieren in een SPF Faciliteit (toestemming ID: SYXK-J-2011-0037). 1. Cmah Knock-out Muis Model <ol><li> Gebruik wildtype C57Bl / 6 muizen van het Comparative Medicine Center van Yangzh...

<ol>
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Het hierin gepresenteerde protocol laat de fenotypische beoordeling van homozygote Cmah- knock-out-muizen toe door de relatieve hoeveelheden Neu5Gc van melk- en levermonsters te analyseren en kwantificeren. De analyse werd uitgevoerd met behulp van een standaard HPLC setup met fluorescentiedetectie. De meest kritische stap van deze procedure is de bereiding van de anionuitwisselingszolen en het uitvoeren van de anionuitwisselingschromatografie; Om de hars goed te regelen en de juiste was- en elueringsf...

N-acetylneuraminezuur (Neu5Ac) en N-glycolylneuraminezuur (Neu5Gc) zijn de meest voorkomende sialinezuren bij de meeste zoogdieren 1 . Hoewel het mogelijk is om Neu5Ac endogeen te synthetiseren, kunnen mensen niet Neu5Gc produceren door een primaire exon deletie op het CMAH gen dat codeert voor een CMP-Neu5Ac hydroxylase 2 , 3 . Dierlijke voedingsmiddelen kunnen echter voedingsbronnen zijn van Neu5Gc 4 , 5 , 6 , wat leidt tot de productie van anti-Neu5Gc antilichamen en leidt daarom tot een immuunrespons tegen Neu5Gc 7 . Dit dieet effect van Neu5Gc wordt vermoedelijk bij te dragen aan chronische ontsteking en diverse andere aandoeningen 8 , 9 , 10 . Om de effecten van Neu5Gc in h te begrijpenUmans, is een diermodel voor de systematische studie van de effecten van voedselgedragen sialicuren zeer wenselijk. Hoewel protocols op basis van polymerase kettingreactie (PCR) voor het analyseren van knock-out muizen goed gevestigd zijn en een handige manier zijn voor de genotypische beoordeling, vereist de functionele analyse van fenotype op het metabolische niveau meer specifieke analysemethoden. Het fenotype van een Cmah knock-out muis model kan worden beoordeeld door het isoleren en analyseren van de samenstelling van sialinezuren in lever- of melkmonsters. Verscheidene methoden voor de detectie van sialinezuren in dierlijke weefsels zijn eerder gemeld: het reageren van sialzuren met resorcinol 11 of thiobarbituriczuur 12 resulteert in de vorming van een chromophorisch product en kan eenvoudig worden geanalyseerd met behulp van een platereader gebaseerde opstelling, maar alleen de totale sialische Zuurgehalte kan worden bepaald. Als alternatief werd ook de analyse van sialinezuren beschreven onder toepassing van gaschromatografie13 , MALDI-ToF-massaspectrometrie 14 of amperometrische methoden 15 . De meest gebruikelijke sialinezuuranalysemethoden zijn echter gebaseerd op hydrolytische afgifte, gevolgd door fluorescentiedivatisering en daaropvolgende vloeibare chromatografie 16 , 17 , 18 met hoge prestaties.

<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 (cytidine monofosfaat-N-acetylneuraminezuurhydroxylase) is verantwoordelijk voor de oxidatie van cytidine monofosfaat-N-acetylneuraminezuren bij zoogdieren. Echter, mensen kunnen cytidine monofosfaat-N-acetylneuraminezuur niet oxideren tot cytidine monofosfaat-N-glycolylneuraminezuur als gevolg van een primaire exon deletie van het CMAH gen. Om de effecten en implicaties van het ontbreken van CMAH-activiteit in meer detail te begrijpen, is een Cmah- knock- outmodel in muizen een grote interesse in basis en toegepast onderzoek. De analysemethode voor het bepalen van het fenotype van dit muismodel wordt hierin gedetailleerd beschreven en is gebaseerd op de detectie van zowel N-acetylneuraminezuur als N-glycolyleenzuurzuur in de lever en melk van wild-type en Cmah- knock- outmuizen . Endogene sialiczuren worden vrijgemaakt en afgeleid met o-fenylendiamine om fluorogene derivaten te genereren, die vervolgens met behulp van HPLC kan worden geanalyseerd. Het gepresenteerde protocol kan zijnOok aangevraagd voor de analyse van melk- en weefselmonsters uit verschillende andere herkomst, en kan van nut zijn om de voedings- en gezondheidseffecten van N-glycolylneuraminezuur te onderzoeken.

Een schematisch overzicht van de beschreven analysemethode is weergegeven in Figuur 1 en omvat de isolatie van sialinezuren uit melk- en levermonsters van wild-type en Cmah- knock-out mutantmuizen en de fluorescentiedivatisering en HPLC-analyse van deze componenten. Figuur 2 en Figuur 3 tonen representatieve HPLC chromatogrammen van afgeleide sialinezuren van melk- en levermons...

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.

We beschrijven een HPLC-gebaseerde methode voor de bepaling van N-acetylneuraminezuur en N-glycolylenuraminezuur in muizenlever en melk.

Bepaling van Sialic Acids in Lever en Melk Monsters van Wild-type en<em&gt; CMAH</em&gt; Knock-out Muizen.

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

Research

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Biology

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

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Quantitation of ÃƒÅ½Ã‚Â³H2AX Foci in Tissue Samples

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

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

In vivo Lever endocytose gevolgd door zuivering van levercellen door leverperfusie

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 ...

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Milk Collection Methods for Mice and Reeves' Muntjac Deer

Melk Collection Methoden voor Mice and Reeves &#39;Muntjac Herten

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

Bepaling van vetzuuroxidatie en Lipogenese in Mouse primaire hepatocyten

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

Melk Collection in de rat met behulp van capillaire buizen en Schatting van melk vetgehalte 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

Filtratie Isolatie van nucleïnezuren: Een eenvoudige en snelle 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 ...