Name: detection of alternative splicing during epithelial-mesenchymal transition
Uploaded: 2014-10-09T14:00:00
Description: Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

1 cellodling av EMT Induktion Anm: EMT kan induceras genom behandling av TGFp eller ektopiskt uttryck av transkriptionsfaktorer Twist, snigel, eller Zeb1 / 2 i epitelceller. Beskrivs i detta protokoll är ett inducerbart EMT system via uttryck av Twist-ER-fusionsprotein i odödliggjorda humana bröstepitelceller (HMLE / Twist-ER, en gåva av Dr J Yang, UCSD) 9,11,29. Vid 4-hydroxitamoxifen (TAM) behandling, translokerar fusionsproteinet Twist-ER till cellkärnan för att driva trans...

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Det förfarande som beskrivs här möjliggör detektion av alternativ splitsning i ett inducerbart EMT modell. Som sådan, dynamiska förändringar av skarv isoform uttryck kan fångas under hela tidsförloppet för EMT. Denna metod har fördelar jämfört med användning av olika epithelial- eller mesenkymala-representerande cellinjer för jämförelse av alternativ splitsning, eftersom olika genetisk bakgrund från un relaterade cellinjer kunde otillbörligt påverka alternativ splitsning. Dock måste den framgångsri...

Det epitel-mesenkymala övergång (EMT) är ett utvecklingsprogram som driver organ morfogenes och vävnadsombildning under embryogenes. När onormalt aktiverad, EMT främjar tumörmetastaser och organfibros 1,2. Övertygande studier har beskrivit betydelsen av transkriptionell reglering under processen för EMT, som definieras av flera transkriptionsfaktorer, såsom Twist, Snail, och ZEB, som undertrycker expressionen av adherens proteinet E-cadherin, vilket resulterar i förlust av en cobble- sten som epitelial morfologi och förstärkning av en spolformad mesenkymala fenotyp 3-8. Nyligen genomförda studier genom genomet hela analysen av RNA visade att det finns en grupp av gener vars skarvning mönster är förknippade med antingen epiteliala eller mesenkymala fenotyper 9,10. Arbeta från vårt labb funktionellt anslutna alternativ splitsning och EMT. Genom att studera cellytan adhesionsmolekylen CD44, visade vi att CD44 alterntivt skarvning är hårt reglerad under EMT, och ännu viktigare, att CD44 skarv isoform byta kausalt bidrar till EMT 11. Alternativ splitsning är en utbredd och bevarad modell av genreglering som upp till 95% av humana multi exon gener alternativt splitsad 12-14. Genom att generera flera proteinprodukter från en enda gen, alternativ splitsning utgör en väsentlig mekanism för protein mångfald, lägga ytterligare ett lager av komplexitet till det mänskliga genomet. Som sådan kunde dysreglering av alternativ splitsning potentiellt leda till djupgående biologiska effekter som orsakar mänskliga sjukdomar. I själva verket har avvikande alternativ splitsning vid sjukdomar har dokumenterats i över ett decennium 15-25, inklusive bedömningar nyligen att mutationer i gener som kodar för spliceosome maskiner är vanliga i myelodysplastiskt syndrom 26-28. Därför utvecklar tillförlitliga metoder för att upptäcka alternativt splitsad isoforms är av stor betydelse i studiet av olika biologiska processer inklusive EMT. Här ger vi ett protokoll för att upptäcka förändringar i alternativ splitsning med hjälp av ett inducerbart EMT modell. Metoderna för att designa PCR-primrar och detektera splits isoformer är lämpliga inte bara för studier av alternativ splitsning under EMT, men också för studier av alternativ splitsning i andra biologiska processer. Undersöka alternativ splitsning under EMT är absolut nödvändigt för att bättre förstå mekanismerna för EMT och tumörmetastaser och därmed underlätta utvecklingen av effektiva strategier för behandling av cancermetastaser.

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			<td height="21" style="height:21px;width:299px;">Name of the reagent</td>
			<td style="width:183px;">Company</td>
			<td style="width:123px;">Catalogue number</td>
			<td style="width:564px;">Comments (optional)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">4-hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>Make stock solution by dissolving in ethanol to 200&#956;M, and keep at -20&#8451; protected from light.&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A. Total RNA Isolation kit</td>
			<td>Omega Bio-Tek</td>
			<td>R6731</td>
			<td>Total RNA isolation kit</td>
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		<tr height="21">
			<td height="21" style="height:21px;">GoScript Reverse Transcription System</td>
			<td>Promega</td>
			<td>A5001</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Real-Time PCR System</td>
			<td>Roche</td>
			<td></td>
			<td>Equipment for qRT-PCR assay</td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD44 antibody</td>
			<td>R&#38;D Systems</td>
			<td>BBA10</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-cadherin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4065</td>
			<td>1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-catenin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2309</td>
			<td>1:1000 dilution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">occludin antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-5562</td>
			<td>1:500 dilution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">fibronectin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610077</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-cadherin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610920</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vimentin antibody</td>
			<td>NeoMarkers</td>
			<td>MS-129-p1</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td>Millipore Corporation</td>
			<td>MAB374</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amasham ECL Western blotting detection reagent</td>
			<td>GE Health Life Science</td>
			<td>RPN2209</td>
			<td>Chemiluminescence system</td>
		</tr>
	</tbody>
</table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

De förfaranden som beskrivs ovan ger en robust metod för att upptäcka alternativ splitsning under EMT. Representativa resultat av CD44 splice isoformen omkoppling under Twist-inducerad EMT ges nedan som exempel. Twist-inducerad EMT i HMLE / Twist-ER-celler kännetecknades av en övergång från en kullersten-sten som epitelial fenotyp till en långsträckt fibroblastrespons fenotyp (Figur 2A), avsaknaden av epiteliala markörer E-cadherin, γ-catenin och occludin och uppr...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Upptäckt av alternativ splits Under Epithelial-Mesenkymala Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Research

JoVE Journal

Biology

Light/dark Transition Test for Mice

Ljus / mörk Transition Test för möss

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Detection of Protein Ubiquitination

Detektion av protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Optimized PCR-based Detection of Mycoplasma

Optimerad PCR-baserad detektion av Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Flera parametrar Mätning av permeabiliteten Transition poröppning i isolerade mus hjärta mitokondrier

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Snabb genetisk analys av Epithelial-mesenkymala Signaling Under Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Induktion och Analys av Epithelial till mesenkymala övergång

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Alternativa kulturer för Human pluripotenta stamceller Produktion, underhåll, samt genetisk analys

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Metoder för att upptäcka alternativa Promoter Användning och transkriptionsreglering av murin Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Ett alternativt tillvägagångssätt för provberedning med låg mobilnummer för TEM-analys

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Molekylär analys av Endothelial-mesenkymala övergången induceras av Transforming Growth Factor-β signalering

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...