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

Dette arbeidet ble delvis støttet av Natural Science Foundation of China (bevilgningsnummer 31471703, A0201300537 og 31671854 til JV og LL), og 100 Foreign Talents Plan (bevilgningsnummer JSB2014012 til JV).

Prosedyrer som involverer dyrefag, er godkjent av Etikkutvalget for Experimentell dyresenter ved Nanjing landbruksuniversitet i samsvar med de nasjonale retningslinjene for dyrevelferd (Vitenskapsministeriet, PR i Kina, 2006) med dyrene som er plassert i en SPF Anlegg (Tillatelses ID: SYXK-J-2011-0037). 1. Cmah Knock-out Mus Modell <ol><li> Bruk wild-type C57Bl / 6-mus fra Comparative Medicine Center of Yangzhou University (Kina). MERK: Cmah knock-out mus ble gener...

<ol>
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Den her presenterte protokollen tillater fenotypisk vurdering av homozygote Cmah- knock-out-mus ved å analysere og kvantifisere de relative mengder av Neu5Gc av melk og leverprøver. Analysen ble utført ved bruk av en standard HPLC-oppsett med fluorescensdeteksjon. Det mest kritiske trinn i denne prosedyren er fremstilling av anionbytterkolonnene og utførelse av anionutvekslingskromatografi; Å avgjøre harpiksen riktig og å samle de riktige vaske- og elueringsfraksjonene tar litt øvelse. <p class="jov...

N-acetylneuraminsyre (Neu5Ac) og N-glykolylneuraminsyre (Neu5Gc) er de vanligste sialinsyrene i de fleste pattedyr 1 . Selv om det er i stand til å syntetisere Neu5Ac endogent, er mennesker ikke i stand til å produsere Neu5Gc på grunn av en primær ekson deletion på CMAH genet som koder for en CMP-Neu5Ac hydroksylase 2 , 3 . Imidlertid kan animalske matvarer være kostholdskilder til Neu5Gc 4 , 5 , 6 , som fører til produksjon av anti-Neu5Gc-antistoffer og dermed utløse en immunrespons mot Neu5Gc 7 . Denne diettvirkningen av Neu5Gc er mistenkt for å bidra til kronisk betennelse og ulike andre sykdommer 8 , 9 , 10 . For å forstå forståelsen av Neu5Gc i hUmans, er en dyremodell for den systematiske studien av virkningene av matbårne sialinsyrer, svært ønskelig. Selv om protokoller basert på polymerasekjedereaksjon (PCR) for analyse av knock-out-mus er veletablert og en praktisk måte for den genotypiske vurderingen, krever funksjonell analyse av fenotype på metabolsk nivå mer spesifikke analysemetoder. Fenotypen av en Cmah- knock-out-musemodell kan vurderes ved å isolere og analysere sammensetningen av sialinsyrer i lever- eller melkeprøver. Flere metoder for påvisning av sialinsyrer i animalsk vev har tidligere blitt rapportert: omsetning av sialinsyrer med resorcinol 11 eller tiobarbitursyre 12 resulterer i dannelsen av et kromoforisk produkt og kan enkelt analyseres ved hjelp av en platereaderbasert oppsett, men bare den totale sialiske Syreinnhold kan bestemmes. Alternativt ble analysen av sialinsyrer også beskrevet under anvendelse av gaskromatografi13 , MALDI-ToF massespektrometri 14 eller amperometriske metoder 15 . Imidlertid er de mest anvendte sialinsyreanalysemetoder basert på hydrolytisk frigjøring, etterfulgt av fluorescensderivatisering og etterfølgende høyytelsesvæskekromatografi 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 (cytidinmonofosfat-N-acetylneuraminsyrehydroksylase) er ansvarlig for oksydasjonen av cytidinmonofosfat-N-acetylneuraminsyrer i pattedyr. Imidlertid kan mennesker ikke oksidere cytidinmonofosfat-N-acetylneuraminsyre til cytidinmonofosfat-N-glykolylneuraminsyre på grunn av en primær exon-deletjon av CMAH- genet. For å forstå effektene og implikasjonene av mangel på CMAH-aktivitet mer detaljert, er en Cmah- knock-out-modell i mus av stor interesse for grunnleggende og anvendt forskning. Analysemetoden for å bestemme fenotypen til denne musemodellen er her beskrevet i detalj, og er basert på deteksjon av både N-acetylneuraminsyre og N-glykolylenuraminsyre i leveren og melk av vill-type og Cmah- knock-out-mus. Endogene sialinsyrer frigjøres og derivatiseres med o-fenylendiamin for å danne fluorogene derivater, som deretter kan analyseres ved HPLC. Den presenterte protokollen kan væreOgså søkt om analyse av melke- og vevsprøver fra forskjellige andre opprinnelser, og kan være nyttig for å undersøke nærings- og helseeffekter av N-glykolylneuraminsyre.

En skjematisk oversikt over den beskrevne analysemetoden er vist i figur 1 og inkluderer isolering av sialinsyrer fra melke- og leverprøver av vildtype- og Cmah- knock-out- mutantmus , og fluorescensderivatiseringen og HPLC-analysen av disse komponentene. Figur 2 og Figur 3 viser representative HPLC-kromatogrammer av derivatiserte sialinsyrer av melke- og leverprøver fra homo- og...

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.

Vi beskriver en HPLC-basert metode for bestemmelse av N-acetylneuraminsyre og N-glykolylenuraminsyre i muselever og melk.

Bestemmelse av sialsyre i lever og melkprøver av villtype og<em&gt; CMAH</em&gt; Knock-out Mus.

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

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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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