Name: visualization of microbiota in tick guts by whole-mount in situ hybridization
Uploaded: 2018-06-01T14:45:00
Description: Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

Vi takke oprigtigt Dr. Mustafa Khokha, Yale University, for at give brugen af hans laboratorium ressourcer. Vi er taknemmelige for Mr. Ming-Jie Wu fremragende teknisk bistand. EF er en HHMI investigator. Dette arbejde blev støttet af en gave fra John Monsky og Jennifer Weis Monsky Lyme Disease Research Fund.

1. forberedelse af DNA skabeloner
<ol><li>Download 16S rRNA sekvens specifikke hver bakteriel slægter fra NCBI database og design Polymerasekædereaktionen (PCR) kompatibel frem og bak primere, der vil forstærke slægter-specifikke regioner (~ 200-250 basepar lang), som beskrevet tidligere 19. også henvise de open source-databaser SILVA20,21 probeBase22 online ressourcer for rRNA-målrettet oligonukleotid sonde...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+set+of+16S+rRNA-based+probes+for+detection+of+bacteria+in+human+feces.">Extensive set of 16S rRNA-based probes for detection of bacteria in human feces.</a> Applied Environmental Microbiology. 68 (6), 2982-2990 (2002).</li><li>Quast, C., 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=The+SILVA+ribosomal+RNA+gene+database+project:+improved+data+processing+and+web-based+tools.">The SILVA ribosomal RNA gene database project: improved data processing and web-based tools.</a> Nucleic Acids Research. 41 (Database issue), D590-D596 (2013).</li><li>Yilmaz, P., 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=The+SILVA+and+&amp;#34;All-species+Living+Tree+Project+(LTP)&amp;#34;+taxonomic+frameworks.">The SILVA and &amp;#34;All-species Living Tree Project (LTP)&amp;#34; taxonomic frameworks.</a> Nucleic Acids Research. 42, D643-D648 (2014).</li><li>Greuter, D., Loy, A., Horn, M., Rattei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=probeBase--an+online+resource+for+rRNA-targeted+oligonucleotide+probes+and+primers:+new+features+2016.">probeBase--an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016.</a> Nucleic Acids Research. 44, D586-D589 (2016).</li><li>Narasimhan, S., 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=Modulation+of+the+tick+gut+milieu+by+a+secreted+tick+protein+favors+Borrelia+burgdorferi+colonization.">Modulation of the tick gut milieu by a secreted tick protein favors Borrelia burgdorferi colonization.</a> Nature Communication. 8 (1), 184 (2017).</li><li>Bryant, S., Manning, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+gel+electrophoresis+of+total+RNA.">Formaldehyde gel electrophoresis of total RNA.</a> Methods Molecular Bioliogy. 86, 69-72 (1998).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98 (2), 81-85 (1989).</li><li>Robson, A., Owens, N. D., Baserga, S. J., Khokha, M. K., Griffin, J. 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Dette er den første brug af en helhed-mount i situ hybridisering (WMISH) teknik til at studere mikrobiota af en leddyr vektor af patogener. Vores protokol blev tilpasset fra en bruges til at undersøge udviklingen i Drosophila og frøen embryoner25,26. Hele-mount RNA i situ hybridisering har været rutinemæssigt anvendes til at lokalisere gen udskrifter rumligt og tidsligt27 og visualisering af udskrifter kan g...

Menneskelige og animalske patogener, der overføres af leddyr vektorer findes over hele verden og tegner sig for omkring 20% af smitsomme sygdomme globalt1, men effektive og sikre vacciner mod de fleste af disse patogener er ikke tilgængelige. Vores forståelse af den vigtige rolle, commensal, symbiotisk og patogene mikroorganismer, kollektivt kendt som microbiome2, i modulerende og udformningen af næsten alle metazoans3 sundhed ekspanderer. Det er nu klart, at leddyr vektorer af patogener også havnen gut mikrobiota og disse vektor-associerede mikrobiota har vist sig at påvirke forskellige vektor-bårne patogener4,5. Leddyr microbiome er sammensat af eubacteria, archaea, vira og eukaryote mikrober såsom protozoer, nematoder og svampe6. Men den fremherskende forskningsfokus har været på eubacteria skyldes, dels at tilgængeligheden af markørgener og reference databaser at identificere specifikke bakteriel medlemmer.
Med fokus på Ixodes scapularis, kryds vektor af flere menneskelige patogener herunder Borrelia burgdorferi7, , det agens af Lyme sygdom, optimering af en mikrobiel visualisering teknik var rettet mod forbedre vores forståelse af kryds gut mikrobiota i forbindelse med vektor-patogen interaktioner. Flere spørgsmål forbliver ubesvaret i feltet kryds microbiome. Tarmen er stedet for den første udvidede møde mellem kryds og indgående patogenet i forbindelse med vandret overføres patogener; Derfor vil forståelse af vektor gut mikrobiota i modulerende vektor-patogen interaktioner rolle afsløre meningsfuld indsigt. Tæger har en enestående tilstand af blodmel fordøjelsen, hvor behandlingen af blodmel komponenter finder sted intracellulært8. Tarm lumen tilsyneladende fungerer som et fartøj indeholder blod måltid som tick feeds, og næringsstof fordøjelse og assimilation opstå i hele flere dage af fodring og fortsætte efter repletion. Patogener erhvervet af kryds under fodring Angiv tarm lumen sammen med blodmel og dermed lumen bliver en primære websted af interaktioner mellem kryds, patogen og bosiddende mikrobiota. Som fordøjelse provenuet gennem repletion og Ixodid kryds molting, gennemgår gut strukturelle og funktionelle ændringer9. Sammensætning og den rumlige organisering af tarm bakterier er også tilbøjelige til at variere i koncert med de skiftende gut milieu. Det er derfor vigtigt at forstå arkitekturen i resident bakterier i kryds tarm til fuldt ud at forstå samspillet mellem kryds, patogen og gut mikrobiota.
Molekylære teknikker til at beskrive vært-associerede mikrobiota rutinemæssigt udnytte høj overførselshastighed parallelle sekventering strategier10 for at forstærke og sekvens bakteriel 16S ribosomale DNA (rDNA). Disse sekventering strategier omgå nødvendigheden af at opnå axenic kulturer af specifikke bakterier og giver en detaljeret beskrivelse af alle bakteriel medlemmer repræsenteret i stikprøven. Sådanne strategier er dog forvirret af manglende evne til at skelne mellem miljømæssige forureninger fra Bonafide beboere. Yderligere, når vurderingen af prøver, som flåter, som er små i størrelse og dermed indeholder lav mikrobiota-specifikke DNA udbytter, sandsynligheden for forstærkning af miljøforurenende er øget11 og resultater i den tvetydige fortolkning af Microbiome sammensætning. Funktionelle karakterisering i forbindelse med visualisering af specifikke levedygtige bakterier vil derfor være afgørende at definere og skelne microbiome af kryds, tidsmæssigt og rumligt. Mod dette mål tog vi fordel af hele-mount RNA i situ hybridisering. Denne teknik bruges rutinemæssigt til at vurdere gen expression mønstre i organer og embryoner12,13,14 og giver mulighed for semikvantitativ analyse af udtryk over hele prøven af interesse. Dette adskiller sig fra traditionelle i situ hybridisering teknikker, der anvender væv sektioner og ofte kræver omfattende analyse af sektioneret materiale med en beregningsmæssige forsamling at forudsige udtryk i hele organer15. Mens hele-mount generelt henviser til hele organismer12, refererer her hele-mount til hele indvolde eller organer. Fordelene ved at bruge hele-mount RNA i situ hybridisering tilgang til at vurdere arkitektur af kryds gut mikrobiota er multifold. Kryds gut er sammensat af 7 par af Divertikler, hvert par varierende i størrelse16. Den funktionelle forskelle, kryds hvis nogen blandt disse Divertikler, ikke er forstået i forbindelse med kryds biologi, mikrobiota eller kryds-patogen interaktioner. Manipulationer af tarmen, at briste gut Divertikler vil fortrænge mikrobiota stede i tarm lumen eller dem løst tilknyttet gut og fører til fejlfortolkning af den geografiske lokalisering af mikrobiota. Fluorescens-mærket RNA i situ hybridisering er blevet udnyttet tidligere for at undersøge kryds gut udskrifter17 ved fastsættelse og åbne individuelle gut diverticula at sikre sonde hybridisering og lokalisere B. burgdorferi udskrifter af skæring paraffin-embedded hele flåter18. Begge disse metoder kræver manipulationer af kryds væv inden hybridisering, der ville påvirke gut mikrobiota arkitekturen.
I denne betænkning beskriver vi i detaljer protokollen for at undersøge levedygtige kryds gut mikrobiota bruger hele-mount i situ hybridisering (WMISH). Brug af hele-mount RNA i situ hybridisering muliggør en global forståelse af tilstedeværelsen og overflod af specifikke tarm bakterier i de forskellige regioner i tarmen og kan anspore til nye indsigter i kryds gut biologi i forbindelse med patogenet kolonisering og transmission. Yderligere, brugen af RNA-sonder rettet mod specifikke bakterielle RNA giver mulighed for påvisning af levedygtige bakterier i kryds gut.

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			<td height="21" style="height:21px;width:320px;">Sefar NITEX Nylon Mesh, 110 micron</td>
			<td style="width:220px;">Amazon</td>
			<td style="width:348px;">03-110/47</td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:320px;">pGEM-T Easy Vector System</td>
			<td style="width:220px;">Promega</td>
			<td style="width:348px;">A1360</td>
			<td></td>
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			<td height="21" style="height:21px;width:320px;">Digoxygenin-11-UTP</td>
			<td style="width:220px;">Roche</td>
			<td align="right" style="width:348px;">1209256910</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">dNTP</td>
			<td style="width:220px;">New England Biolabs&#160;</td>
			<td style="width:348px;">N0447S</td>
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			<td height="21" style="height:21px;width:320px;">DNAse I(RNAse-free)</td>
			<td style="width:220px;">New England Biolabs</td>
			<td style="width:348px;">M0303S</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">HiScribe SP6 RNA synthesis kit</td>
			<td style="width:220px;">New England Biolabs</td>
			<td style="width:348px;">E2070S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">HiScribe T7 High Yield RNA Synthesis Kit</td>
			<td style="width:220px;">New England Biolabs</td>
			<td style="width:348px;">E2040S</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Water, RNase-free, DEPC-treated</td>
			<td style="width:220px;">American Bioanalytical</td>
			<td style="width:348px;">AB02128-00500</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">EDTA, 0.5M, pH 8.0</td>
			<td style="width:220px;">American Bioanalytical</td>
			<td style="width:348px;">AB00502-01000</td>
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			<td height="21" style="height:21px;width:320px;">Formaldehyde, 37%</td>
			<td style="width:220px;">JT Baker</td>
			<td style="width:348px;">2106-01</td>
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			<td height="21" style="height:21px;width:320px;">Formamide</td>
			<td style="width:220px;">American Bioanalytical</td>
			<td style="width:348px;">AB00600-00500</td>
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			<td height="21" style="height:21px;width:320px;">EGTA</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">E-4378</td>
			<td></td>
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			<td height="21" style="height:21px;width:320px;">DPBS, 10X</td>
			<td style="width:220px;">Gibco</td>
			<td style="width:348px;">14300-075</td>
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			<td height="21" style="height:21px;width:320px;">Tween-20</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">P1379-25ML</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Proteinase K</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">3115879001</td>
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			<td height="21" style="height:21px;width:320px;">Triethanolamine HCl</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">T1502-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Acetic anhydride</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">320102-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Paraformaldehyde</td>
			<td style="width:220px;">ThermoScientific/Pierce</td>
			<td align="right" style="width:348px;">28906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">SSC, 20X</td>
			<td style="width:220px;">American Bioanalytical</td>
			<td style="width:348px;">AB13156-01000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RNA from torula yeast</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">R3629-5G</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Heparin, sodium salt</td>
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			<td style="width:348px;">H3393-10KU</td>
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			<td height="21" style="height:21px;width:320px;">Denhardt&#39;s Solution, 50X</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">D2532-5ML</td>
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			<td height="21" style="height:21px;width:320px;">CHAPS hydrate</td>
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			<td height="21" style="height:21px;width:320px;">RNase A</td>
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			<td align="right" style="width:348px;">10109142001</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RNase T1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Maleic acid</td>
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			<td height="21" style="height:21px;width:320px;">Blocking reagent</td>
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			<td align="right" style="width:348px;">11096176001</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-Digoxigenin-AP, Fab fragments</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td align="right" style="width:348px;">11093274910</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Levamisol hydrochloride</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">31742-250MG</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:320px;">Chromogenic substrate for alkaline phosphatase</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bouin&#39;s solution</td>
			<td style="width:220px;">Sigma Aldrich</td>
			<td style="width:348px;">HT10132-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hydrogen peroxide</td>
			<td style="width:220px;">Mallinkrodt Baker, Inc</td>
			<td style="width:348px;">2186-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Single stranded RNA ladder</td>
			<td style="width:220px;">Ambion -Millenium</td>
			<td style="width:348px;">AM7151</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;#11 High-Carbon steel blades&#160;</td>
			<td style="width:220px;">C and A Scientific Premiere</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Thermocycler</td>
			<td style="width:220px;">BioRad, CA</td>
			<td align="right" style="width:348px;">1851148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Spectrophotometer</td>
			<td style="width:220px;">ThermoScientific&#160;</td>
			<td style="width:348px;">NanoDrop 2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Orbital shaker</td>
			<td style="width:220px;">VWR</td>
			<td style="width:348px;">DS-500E Digital Orbital shaker</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Shaking water bath</td>
			<td style="width:220px;">BELLCO Glass, Inc</td>
			<td style="width:348px;">Hot Shaker-7746-12110</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Gel&#160; documentation system</td>
			<td style="width:220px;">BioRad</td>
			<td style="width:348px;">Gel Doc XR+ Gel documentation system</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Nikon</td>
			<td style="width:348px;">NikonSM2745T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bright-field Microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">AXIO Scope.A1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dissection microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">STEMI 2000-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Light box</td>
			<td style="width:220px;">VWR</td>
			<td style="width:348px;">102097-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">PCR purification kit&#160;</td>
			<td style="width:220px;">Qiagen</td>
			<td align="right" style="width:348px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image capture software</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:348px;">Zen lite</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image editing software&#160;</td>
			<td style="width:220px;">Adobe&#160;</td>
			<td style="width:348px;">Adobe Photoshop CS4 version 11.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Image analysis software</td>
			<td style="width:220px;">National Institutes of Health</td>
			<td>ImageJ-NIH /imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;">Automation compatible instrumentation</td>
			<td style="width:220px;">Intavis Bioanalytical Instruments, Tubingen, Germany).&#160;</td>
			<td style="width:348px;">Intavis, Biolane HT1.16v</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of microbiota in tick guts by whole-mount in situ hybridization

Smitsomme sygdomme, der overføres af leddyr vektorer fortsætte med at udgøre en væsentlig trussel mod menneskers sundhed på verdensplan. De patogener, der forårsager disse sygdomme, eksisterer ikke isoleret, når de kolonisere vektor; snarere, de sandsynligvis engagere sig i interaktioner med hjemmehørende mikroorganismer i tarm lumen. Vektor mikrobiota har vist sig for at spille en vigtig rolle i patogen transmission for flere vektor-bårne sygdomme. Om bopæl bakterier i tarmen af Ixodes scapularis kryds, vektor af flere menneskelige patogener herunder Borrelia burgdorferi, påvirke kryds transmission af patogener er ikke fastlagt. Vi kræver metoder for kendetegner sammensætningen af bakterier forbundet med kryds tarmen til at fremme en bedre forståelse af potentielle interspecies interaktioner i kryds gut. Ved hjælp af hele-mount i situ hybridisering til at visualisere RNA udskrifter tilknyttet særlige bakteriearter giver mulighed for indsamling af kvalitative data med hensyn til forekomsten og fordelingen af mikrobiota i intakt væv. Denne teknik kan bruges til at undersøge ændringer i gut mikrobiota milieu i løbet af kryds fodring og kan også anvendes til at analysere udtryk for kryds gener. Farvning af hele kryds indvolde udbytte oplysninger om grov rumlige fordeling af target RNA i vævet uden behov for tre-dimensionelle genopbygning og påvirkes mindre af miljøforurening, der ofte tilintetgør sekventering-baseret metoder ofte brugt til at studere komplekse mikrobielle samfund. Samlet set er denne teknik et værdifuldt redskab, der kan bruges til bedre forstå vektor-patogen-mikrobiota interaktioner og deres rolle i smitteoverførsel.

Måling og vurdering af kvaliteten af RNA-sonder er kritiske forud for begyndelsen farvning. In vitro transskription effektivitet afhænger meget af mængden og kvaliteten af DNA-template. Vi visualiseret rutinemæssigt RNA-sonder på en formaldehyd gel til at kontrollere renhed og mængden af sonden genereret af transskription reaktioner. Sonderne bør fremstå som lyse, diskrete bands (figur 2). Spektrofotometriske målinger af RNA-koncentrationen ...

Watch this Scientific Journal Video about Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization at JoVE.com

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Visualisering af mikrobiota i kryds tarme af hele-mount In Situ hybridisering

Vi præsenterer her, en protokol for at rumligt og tidsligt vurdere tilstedeværelsen af levedygtige mikrobiota i kryds modet ved hjælp af en modificeret hele-mount in situ hybridisering tilgang.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Research

JoVE Journal

Genetics

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

In Situ Mærkning af mitokondrie-DNA Replication i Drosophila Adult Ovarier af Edu Farvning

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Tilfældig mutagenese ved anvendelse histon-miniSOG i<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

Påvisning af Inter-kromosomale Stabil Afvigelser fra Multiple Fluorescens<em&gt; In Situ</em&gt; Hybridisering (MFISH) og Spectral Karyotyping (SKY) i bestrålede mus

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Målrettet<em&gt; I Situ</em&gt; Mutagenese af histon-gener i knopskydende gær

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

Høj opløsning fluorescerende In Situ hybridisering i Drosophila fostre og væv ved hjælp af Tyramide Signal forstærkning

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

Enkelt molekyle fluorescens In Situ hybridisering (smFISH) analyser i spirende gær vegetativ vækst og meiose

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Kvantitativ analyse af Alternative Pre-mRNA-Splicing i mus hjernen sektioner ved hjælp af RNA In Situ hybridisering Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Væv-specifikke miRNA udtryk profilering i mus hjerte sektioner ved hjælp af In Situ hybridisering

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

At hæve den mexicanske Tetra Astyanax mexicanus for analyse af Post-larval fænotyper og hele-mount Immunohistokemi

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Kendetegner Histon posttranslationel modifikation ændringer i gær Neurodegenerative Proteinopathy modeller

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...