Name: 3d multicolor dna fish tool to study nuclear architecture in human primary cells
Uploaded: 2020-01-25T13:00:00
Description: Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

Forfatterne anerkender den tekniske bistand fra INGM Imaging Facility (Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" (INGM), Milano, Italien), især C. Cordiglieri, for at få hjælp under 3D flerfarvet DNA FISH billeder Erhvervelse. Dette arbejde er blevet støttet af følgende tilskud til B.B.: EPIGEN italiensk flagskibsprogram, Association Française contre les Myopaties (AFM-Telethon, grant nr 18754) og Giovani Ricercatori, det italienske sundhedsministerium (GR-2011-02349383). Dette arbejde er blevet støttet af følgende tilskud til F.M.: Fondazione Cariplo (Bando Giovani, tilskud nr 2018-0321).

1. DNA-sonde forberedelse og mærkning procedurer med nick oversættelse
<ol><li>Purify og ren bakterielle kunstige kromosomer (BaCs), plasmider eller PCR produkter med specifikke kits(Tabel over materialer),resuspend i ddH2O, kontrollere ved elektroforese på en agarose gel og kvantificere.</li>
	<li>Udfør nick oversættelse på 1,5−2 μg DNA fra trin 1.1 i et slutvolumen på 50 μL ved at blande alle reagenser i et 0,5 ml lavt bindende DNA-rør i henhold til tabel 1.<br...

<ol>
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H., 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=Extensive+Heterogeneity+and+Intrinsic+Variation+in+Spatial+Genome+Organization.">Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization.</a> Cell. 176 (6), 1502-1515 (2019).</li><li>Zheng, M., 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=Multiplex+chromatin+interactions+with+single-molecule+precision.">Multiplex chromatin interactions with single-molecule precision.</a> Nature. 566 (7745), 558-562 (2019).</li><li>Solovei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH)+on+tissue+cryosections.">Fluorescence in situ hybridization (FISH) on tissue cryosections.</a> Methods in Molecular Biology. 659, 71-82 (2010).</li><li>Cremer, M., 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=Multicolor+3D+fluorescence+in+situ+hybridization+for+imaging+interphase+chromosomes.">Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.</a> Methods in Molecular Biology. 463, 205-239 (2008).</li><li>Legant, W. 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Den nuværende metode beskriver en trin for trin-protokol til at udføre 3D multicolor DNA FISH på en bred vifte af menneskelige primære celler. Selv om DNA FISH er en teknologi i bred anvendelse, 3D flerfarvet DNA FISK på bevaret 3D interfasede kerner er stadig vanskeligt at udføre i mange laboratorier, hovedsagelig på grund af de særlige kendetegn ved de anvendte prøver23,24.
Sonde nick oversættelse er et grundlæggende skrid...

Højere eukaryoter nødt til systematisk at kondensere og komprimere en enorm mængde af genetiske oplysninger i det øjeblik 3D plads i kernen1,2,3,4. I dag ved vi, at genomet er rumligt bestilt i rum og topologisk tilknyttede domæner5, og at de mange niveauer af DNA foldning generere kontakter mellem forskellige genomiske regioner, der kan involvere kromatin loop dannelse6,7. 3D dynamisk looping af kromatin kan påvirke mange forskellige biologiske processer såsom transskription8,9,differentiering og udvikling10,11, DNA reparation12,13, mens dens forstyrrelser er involveret i forskellige sygdomme14,15,16 og udviklingsmæssige defekter15,17,18.
Mange tilgange er blevet udviklet til at studere 3D genom organisation. Kromosom konformation capture-baserede teknologier (C-teknologier, 3C, 4C, 5C, Hi-C og derivater) er blevet udviklet til at studere genom organisation i faste celler3,4,19,20. Sådanne tilgange er baseret på evnen til at fange kontaktfrekvenser mellem genomiske loci i fysisk nærhed. C-teknologier, afhængigt af deres kompleksitet, fange den globale 3D genom organisation og nuklear topologi af en celle befolkning3,4,19,20. Ikke desto mindre, 3D-interaktioner er dynamiske i tid og rum, meget variabel mellem de enkelte celler, der består af multiplex interaktioner, og er meget heterogen21,22.
3D multicolor DNA fluorescens in situ hybridization (FISH) er en teknik, der gør det muligt at visualisering af specifikke genomiske loci på et enkelt celleniveau, der muliggør direkte undersøgelse af 3D-nuklear arkitektur på en supplerende måde til C-teknologier. Det repræsenterer en teknologi, der i øjeblikket anvendes til entydigt at validere C-resultater. 3D flerfarvet DNA FISH bruger fluorescerende mærket sonder supplerer den genomiske loci af interesser. Brugen af forskellige fluorophorer og egnet mikroskopiudstyr gør det muligt at visualisering af flere mål inden for det nukleare rum23,24. I de senere år er FISH blevet kombineret med teknologiske fremskridt inden for mikroskopi for at opnå visualisering af fine strukturer ved høj opløsning25,26 eller med CRISPR-Cas tilgange til visualisering af nukleinsyrer i levende billeddannelse27,28. På trods af bred vedtagelse, 3D multicolor DNA FISH tilgang er stadig betragtes som vanskeligt i mange laboratorier, fordi det biologiske materiale, der anvendes skal tilpasses.
Her leverer vi en omfattende protokol for 3D multicolor DNA FISH (fra celle / sonde forberedelse til dataanalyse), der gælder for en bred vifte af menneskelige primære celler, så visualisering af flere genomiske loci og bevare 3D-strukturen af kerner. For at studere nuklear arkitektur skal 3D-strukturen af kerner bevares. Derfor undgår vi i modsætning til andre eksisterende protokoller29,30,31brugen af en alkoholgradient og opbevaring af dæksedler i alkohol, der kan påvirke kromatinstruktur32. Metoden er tilpasset fra bevarede 3D DNA FISH protokoller24,33, der skal anvendes på en bred vifte af menneskelige primære celler, både isolerede ex vivo eller dyrket in vitro. Der er permeabiliserings- og deproteiniseringsparametre for forskellige nukleare morfologi og cytologiske egenskaber (f.eks. forskellige grader af nuklear komprimering, cytoskeletoverflod)34. Disse parametre er ofte generelt beskrevet i andre protokol24,33, uden at give en klar forskelsbehandling af proceduren inden for forskellige celletyper. Desuden har vi udviklet et specifikt værktøj ved navn NuCLεD (nukleare kontakter locator i 3D)16, der giver principper for dataanalyse, der vil forbedre 3D nærhed mellem forskellige loci og deres nukleare topologiske distribution inden for det nukleare rum på en automatiseret måde.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin 488</td>
			<td>DBA</td>
			<td>DI7488</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin 11 d-UTP</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0081</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumine)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverlsips</td>
			<td>Marienfeld</td>
			<td>117500</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 d-UTP</td>
			<td>GE Healthcare</td>
			<td>PA53022</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D21490</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acids single strand from salmon testes</td>
			<td>Sigma</td>
			<td>D7656</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran sulfate (powder)</td>
			<td>Santa Cruz</td>
			<td>sc-203917A</td>
			<td></td>
		</tr>
		<tr>
			<td>Digoxigenin 11 d-UTP</td>
			<td>Roche</td>
			<td>11093088910</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>21969-035 500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>18010-017</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>AMPD1</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (C-G-A-T)</td>
			<td>Euroclone</td>
			<td>BL0423A/C/G</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>E9644.2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>02860-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Hyclone</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30109</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F8775-25mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutammine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>30721</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15279-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Human</td>
			<td>Sigma</td>
			<td>I9278-5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>NaAc (Sodium Acetate, pH 5.2, 3 M)</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate-buffered saline)</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennycillin/Streptavidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070-063 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Biorad</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>PhasePrep BAC DNA Kit</td>
			<td>Sigma</td>
			<td>NA0100-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Gel Extraction &#38; PCR Purification Combo Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K220001</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink Quick Plasmid Miniprep Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K210010</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2288</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubbercement</td>
			<td>Bostik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>631-0114</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidina Alexa fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>S21374</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Sodium Citrate</td>
			<td>Sigma</td>
			<td>1110379026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P9416-100mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d multicolor dna fish tool to study nuclear architecture in human primary cells

Et vigtigt spørgsmål i cellebiologi er genomisk organisation inden for det nukleare rum, og hvordan kromatinarkitektur kan påvirke processer som genekspression, celleidentitet og differentiering. Mange tilgange udviklet til at studere 3D-arkitekturen af genomet kan opdeles i to komplementære kategorier: kromosom kropsbygning capture baseret teknologier (C-teknologier) og billeddannelse. Mens førstnævnte er baseret på at fange de kromosom reformation og proksimale DNA interaktioner i en population af faste celler, sidstnævnte, baseret på DNA fluorescens in situ hybridization (FISH) på 3D-bevarede kerner, giver mulighed for moderne visualisering af multipel loci på et enkelt celleniveau (multifarve), undersøge deres interaktioner og fordeling i kernen (3D multicolor DNA FISH). Teknikken med 3D multicolor DNA FISH har en begrænsning af visualisere kun et par forudbestemt loci, ikke tillader en omfattende analyse af den nukleare arkitektur. I betragtning af resultaternes robusthed er 3D-flerfarvet DNA-FISK i kombination med 3D-mikroskopi og billedrekonstruktion imidlertid en mulig metode til validering af C-teknologibaserede resultater og utvetydigt undersøge placeringen og tilrettelæggelsen af specifikke på et enkelt celleniveau. Her foreslår vi en trin for trin metode til 3D multicolor DNA FISK egnet til en bred vifte af menneskelige primære celler og diskutere alle de praktiske handlinger, afgørende skridt, forestillinger om 3D-billedbehandling og dataanalyse er nødvendige for at opnå en vellykket og informativ 3D multicolor DNA FISK i forskellige biologiske sammenhænge.

Metoden til 3D multicolor DNA FISH beskrevet i denne artikel giver mulighed for moderne visualisering af forskellige genomiske loci inden bevaret 3D kerner (Figur 1B). Denne protokol gør det muligt at måle afstande mellem alleler og forskellige genomiske loci er for at vurdere deres rumlige nærhed og vurdere deres placering i det nukleare rum (f.eks. loci afstand fra centroid eller periferien af kerner)16. Der er dog mange afgørende trin, der skal...

Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

3D multicolor DNA FISH repræsenterer et værktøj til at visualisere flere genomiske loci inden for 3D bevaret kerner, utvetydigt definere deres gensidige interaktioner og lokalisering i det nukleare rum på et enkelt celleniveau. Her beskrives en trinvis protokol for et bredt spektrum af humane primære celler.

3D Multicolor DNA FISH Tool til at studere nuklear arkitektur i humane primære celler

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

3D flerfarvede DNA FISH gør det muligt at visualisere flere genomiske loci inden for bevarede kerner for tvetydigt at definere deres gensidige interaktion og lokalisering på enkeltcelleniveau. 3D flerfarvet DNA FISH giver mulighed for direkte undersøgelse af den nukleare arkitektur. Generelt fungerer det sammen med kromosomopsamlingsbaserede teknologier, der gør teknikken til et tilgængeligt værktøj til C-datavalidering i enkelte celler.Demonstration af proceduren vil være Federica Marasca. Hun er postdoc i mit laboratorium. Begynd med at inkubere nick oversættelse mix i en termisk mixer ved 16 grader Celsius i henhold til længden af start-DNA-materiale.Ved slutningen af inkubationen kontrolleres størrelsen af sonderne på en 2,2% agarosegel. For hvert DNA FISH-forsøg udfældes den passende mængde sonde i henhold til det udgangs-DNA-materiale, hvorfra sonderne blev fremstillet i 150 mikroliter dobbeltdestilleret vand 20 mikrogram umærket laks sperm DNA, 3,5 mikrogram af arter specifikke Cot-1 DNA, tre mængder af 100% ethanol, og en 1/10th volumen af tre molar natriumacetat i en time ved minus 80 grader Celsius. Ved inkubationens afslutning centrifugeres prøven ved maksimal hastighed i en time ved fire grader Celsius.Efter udsmid af supernatanten vaskes pellet to gange med 500 mikroliter på 70% ethanol. Efter den anden vask, resuspend pellet i to mikroliter på 100% formamid og ryst sonden i 30 minutter ved 40 grader Celsius, før du tilføjer et tilsvarende volumen på 4X saltvand natriumcitrat i 20% dextran sulfat. Til fiksering forbehandling og permeabilisering af små celler med små kerner og en lav mængde cytoplasma, først tilføjes to gange 10 til de sjette celler i 200 mikroliter PBS direkte på glas coverslips i individuelle brønde af en 24-brønd plade og lad cellerne til at bosætte sig i 30 minutter ved stuetemperatur.Ved inkubationens afslutning udskiftes PBS hurtigt med 300 mikroliter frisklavet 4% paraformaldehyd suppleret med 0,1% Tween 20. Efter 10 minutter vaskes de faste celler tre gange i 300 mikroliter på 0,05% TPBS i fem minutter pr. vask ved stuetemperatur. Efter den sidste vask, permeabilize T-cellerne med 300 mikroliter på 0,5% TPBS i 10 minutter ved stuetemperatur, før du behandler cellerne med 250 mikroliter pr brønd af RNAse cocktail fortyndet 1:100 i PBS i en time ved 37 grader Celsius.Ved slutningen af inkubationen skylles cellerne i 300 mikroliter af PBS og der tilsættes 300 mikroliter på 20% glycerol i PBS til hver brønd med en overnight inkubation ved fire grader Celsius. Næste morgen skal glasset lægges på tøris i 15 til 30 sekunder for at fryse cellerne, før prøverne gradvist optøes ved stuetemperatur og iblødsætning i 300 mikroliter på 20% glycerol i PBS i 30 sekunder. Efter indfrysning/optøning af cellerne tre gange mere som blot påvist, vaskes prøverne i 300 mikroliter på 0,5% TPBS i fem minutter ved stuetemperatur efterfulgt af to vaske i 300 mikroliter på 0,05% TPBS i fem minutter pr. vask ved stuetemperatur.Efter den sidste vask behandles cellerne med 300 mikroliter af 0,1 normal saltsyre i 12 minutter ved stuetemperatur efterfulgt af to skylninger i 300 mikroliter saltvandsnatriumcitrat. Derefter fastgør prøverne natten over i 300 mikroliter på 50% formamid i 2X saltvand natriumcitrat ved stuetemperatur. Til fiksering, forbehandling og permeabilisering af store celler med en høj mængde cytoplasma, fastgør, forbehandling og permeabilisere cellerne på samme måde som påvist for humane primære T-celler og behandle prøverne med 300 mikroliter på 0,0025% pepsin i 0,01 normal saltsyre i et par sekunder op til fem minutter.Observere cellerne under et optisk mikroskop og stoppe reaktionen med to fem minutters vaske i 300 mikroliter af 50 millimolar magnesiumchlorid, så snart kernerne er fri for cytoplasmaet, men nucleoli er stadig synlige og intakte. For 3D flerfarvet DNA FISH, afkoste hybridisering sonder ved 80 grader Celsius i fem minutter og straks placere sonder på is. Læg sonderne på et rent mikroskopskreds, og placer en dækslæb af celler på hver dråbe sonde.Seal dækslædkanterne med gummicement. Når cementen er tørret helt, protese prøverne på en 75 grader Celsius varmeblok. Efter fire minutter, hydridize prøverne ved 37 grader Celsius natten over i en metallisk kasse flyder i et vandbad.Den næste morgen, skalle gummi cement og med dias nedsænket i 2X saltvand natriumcitrat forsigtigt løfte ud coverslips. Placer hver coverslip i individuelle brønde af en seks-brønd plade, der indeholder to milliliter frisk saltvand natriumcitrat pr brønd i to fem minutters vask ved 37 grader Celsius med omrystning ved 90 omdrejninger i minuttet. Ved inkubationens afslutning skylles dækslerne kortvarigt i to milliliter på 0,2% Tween 20 i 4X saltvandsnatriumcitrat.For 3D flerfarvet DNA FISH detektion, overføre coverslips i individuelle brønde af en ny 24-brønd plade og blokere enhver uspecifik binding i 300 mikroliter af blokering buffer i 20 minutter ved 37 grader Celsius og 20 omdrejninger i minuttet. Ved inkubationens afslutning behandles prøverne med en passende koncentration af anti-digoxigenin og/eller streptavidin fortyndet i blokerende buffer i 35 minutter i et mørkt og vådt kammer ved 37 grader Celsius. Ved inkubationens afslutning overføres prøverne til individuelle brønde af en 6-brøndplade, og prøverne vaskes tre gange i to milliliter på 0,2 % Tween 20 og 4X saltvandsnatriumcitrat i fem minutter pr. vask ved 90 omdrejninger i minuttet.Efter den sidste vask skal prøverne bringes i ligevægt i to milliliter PBS, inden dækslerne overføres til en ny 24-brøndplade til efterfastsættelse i 300 mikroliter på 2% formaldehyd i PBS pr. brønd i to minutter ved stuetemperatur. De faste celler vaskes fem gange kortvarigt i 300 mikroliter PBS efterfulgt af farvning med DAPI fortyndet ved et nanogram pr. milliliter i 300 mikroliter PBS i fem minutter ved stuetemperatur. Dækslerne vaskes fem gange mere i PBS, og prøverne monteres med et passende monteringsmedium.Derefter erhverve 3D-billeder med et mikroskop system. 3D flerfarvet DNA FISH giver mulighed for moderne visualisering af forskellige genomiske loci inden for bevaret 3D kerner. Til fremstilling af DNA-sonde sikrer en sondestørrelse på mindre end 200 basispar en vellykket 3D flerfarvet DNA FISH-procedure.Suboptimal DNA FISH sonder produceret af nick oversættelse kan være helt eller delvist fordøjet. Over fordøjede sonder vil resultere i et uspecifikt signal på grund af tab af specificitet i hybridiseringen og en deraf følgende forøgelse af baggrunden. For humane primære T-lymfocytter og små celler med små kerner og en lav mængde cytoplasma anbefales en normal 0,1 normal saltsyrebehandling på 12 minutter for at fremme kerneadgang til DNA-sonderne og bevare den nukleare integritet.Cytoplasmatisk pepsin fordøjelse er ikke nødvendig for at opnå en DNA FISH godt signal i små celler. For menneskelige primære myoblaster og celler, der har store kerner og rigelige cytoplasma, dog, pepsinization trin er grundlæggende. En kort og suboptimal cytoskeleton pepsinisering vil hæmme indtræden af sonden i kernerne slutter i mangel af en DNA FISH signal.Men hvis cellerne er over pepsinized, kerner vil ikke forblive intakt miste deres 3D-struktur. En vellykket 3D flerfarvet DNA FISH vil resultere i tilstedeværelse af signal i kernerne. Jeg foreslår at arbejde med frisk biologisk materiale, teste sonder, før du udfører 3D flerfarvet DNA FISH, forberede friske løsninger og være præcis med inkubation gange og temperaturer.Husk, at formamid og HCL er farlige stoffer og altid bør anvendes i en kemisk røghætte med passende engangsmaterialer.

3d-multicolor-dna-fish-tool-to-study-nuclear-architecture-human

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Parallel Måling af døgnrytmen ur genekspression og hormon sekretion i Human primære cellekulturer

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Tekniske kunstige faktorer til specifikt manipulation af alternativ splejsning i humane celler

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

Laser Microirradiation at studere In Vivo cellulære svar til Simple og komplekse DNA-skader

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

Immunfarvning for DNA ændringer: beregningsmæssige analyse af Konfokal billeder

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

ATAC-seq Assay med lav mitokondriel DNA forurening fra primære menneskelige CD4 + T-lymfocytter

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

Forbedret gær One-hybrid skærme til at identificere transskription faktor Binding til menneskelige DNA-sekvenser

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are then&#160;used to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

Multi-locus variabel-tal tandem-Gentag analyse af den fisk-patogene bakterie Yersinia ruckeri ved MULTIPLEX PCR og kapillar elektroforese

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Navigering MARRVEL, et webbaseret værktøj, der integrerer menneskelig Genomics og model organisme genetik information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

In vivo funktionel undersøgelse af sygdoms associerede sjældne humane varianter ved hjælp af Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Karakterisering af In Vitro Differentiering af humane primære Keratinocytter ved RNA-Seq Analyse

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...