Name: live cell imaging of chromosome segregation during mitosis
Uploaded: 2018-03-14T15:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

Vi vil gerne takke Dr. Guo-Chiuan Hung og Dr. Bharatkumar Joshi for værdifulde kommentarer, at forbedret håndskriftet.

Alle eksperimenter udført i disse studier blev udført i overensstemmelse med protokoller godkendt af institutionelle Animal Care og brug Udvalget på forskningsfacilitet Food and Drug Administration (FDA).
1. isolering og kultur af musen embryonale fibroblaster (MEFs)
<ol><li>Isolere mus embryonale fibroblaster (MEFs) fra en PP2A-B56γ-mus stamme og vildtype littermates af standardprotokol 7,8,9.<...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle+assembly+checkpoint.">The spindle assembly checkpoint.</a> Curr Biol. 22, 966-980 (2012).</li><li>Jallepalli, P. V., Lengauer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+segregation+and+cancer:+cutting+through+the+mystery.">Chromosome segregation and cancer: cutting through the mystery.</a> Nat Rev Cancer. (2), 109-117 (2001).</li><li>Zhu, L., 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=Mitotic+protein+CSPP1+interacts+with+CENP-H+protein+to+coordinate+accurate+chromosome+oscillation+in+mitosis.">Mitotic protein CSPP1 interacts with CENP-H protein to coordinate accurate chromosome oscillation in mitosis.</a> J Biol Chem. 290 (45), 27053-27066 (2015).</li><li>Pikor, L., Thu, K., Vucic, E., Lam, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+detection+and+implication+of+genome+instability+in+cancer.">The detection and implication of genome instability in cancer.</a> Cancer Metastasis Rev. 32 (3-4), 341-352 (2013).</li><li>Varadkar, P., Despres, D., Kraman, M., Lozier, J., Phadke, A., Nagaraju, K., McCright, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+phosphatase+2A+B56&#947;+regulatory+subunit+is+required+for+heart+development.">The protein phosphatase 2A B56&#947; regulatory subunit is required for heart development.</a> Dev Dyn. 243 (6), 778-790 (2014).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Mouse+Embryonic+Fibroblast+Cells+Suitable+for+Culturing+Human+Embryonic+and+Induced+Pluripotent+Stem+Cells.">Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells.</a> J. 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Cellecyklus kontrol checkpoints, der sikrer nøjagtig kromosom segregation forhindre aneuploidi og celle transformation 1,2,3. I den foreliggende undersøgelse, fandt vi at inaktivering af PP2A-B56γ resulterede i en svækket spindel forsamling checkpoint. Levende celle imaging tillod os at observere kromosomale mis segregation under mitosen i PP2A-B56γ MEFs behandlet med nocodazole 9.
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I den følgende protokol beskriver vi en praktisk metode til at visualisere den kromosomale segregation og mitotiske progression i løbet af celle cyklus i mus embryonale fibroblaster ved hjælp af Histon 2B-normal god landbrugspraksis, BacMam 2.0 mærkning og levende celle imaging.
Cellecyklus kontrol checkpoints overvåge kromosom segregation og spiller en vigtig rolle i opretholdelsen af den genetiske integritet i celle 1,2,3. Ophobning af mis segregeret kromosomer kan føre til aneuploidi, som er kendetegnende for mest solide tumorer 4. Derfor, påvisning af halter kromosomer kan bruges som en metode til at studere kromosomale ustabilitet.
Fluorescently mærket proteiner kan blive brugt til at visualisere live kromosom segregation og kromosomale halter men generation af mCherry-mærkede eller H2B-NGL markeret protein kræver betydelig viden om gen levering og molekylærbiologi 5. Her beskrives brug af CellLight Histone2B-NGL BacMam 2.0 reagens, herefter kaldet CL-HB regent, for nemheds skyld. Dette reagens kan anvendes umiddelbart og dermed eliminerer bekymringer om vektor kvalitet og integritet. Derudover kræver dette reagens ikke brug af potentielt skadelige behandlinger eller lipider og dye-loading kemikalier. I modsætning til konventionelle fluorescerende etiketter, CL-HB regent pletter uafhængigt af funktion (dvs., membran potentiale). CL-HB regent kan være blot tilføjet til cellerne og inkuberes natten over i protein udtryk. CL-HB regent replikere ikke i pattedyrceller og kan bruges i biosikkerhed (BSL) 1 laboratorium indstillinger. Også, dette forbigående Transfektion kan påvises efter natten inkubation i op til 5 dage, tid nok til at udføre mest dynamiske cellulære analyser.
Alternativt, kromosomafvigelser kunne studeres ved hjælp af forskellige teknikker såsom flowcytometri, immunhistokemi eller fluorescens i situ hybridisering (FISH) 6. Flowcytometri kan bruges til at studere aneuploidi, som kan måles baseret på DNA indhold og fasen af celler i celle cyklus. Selvom flowcytometri kan bruges til at måle aneuploidi, det ikke give oplysninger om kromosomale mis adskillelse. FISK og Immunhistokemi teknikker bruge fluorescerende sonder til at binde til DNA eller kromosomer. Mens disse teknikker giver et øjebliksbillede af status af en population af celler, tillader de ikke levende celle imaging dermed mangler oplysninger indhentet gennem spatiotemporelle visualisering af cytokinesis i bestemte celler fulgt over en periode.
Denne protokol blev brugt til at studere halter kromosomer eller kromosomal mis adskillelse i nocodazole behandlede mus embryonale fibroblaster (MEFs) isoleret fra PP2A-B56γ-mus. I tillæg til ovenstående program giver denne protokol et simpelt værktøj til at mærke og visualisere kromosomale adskillelse i forskellige celletyper, som kan bruges til at studere celle cyklus regulering eller kromosomal ustabilitet i tumorceller. Det kan desuden også bruges til at studere kromosomale ustabilitet forårsaget af forskellige medicinske behandlingsmetoder eller at studere virkningerne af genet knock ud resulterer i kromosomale mis adskillelse.

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			<td height="21" style="height:21px;width:191px;">DMEM/F12</td>
			<td style="width:135px;">Gibco</td>
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			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Gibco</td>
			<td align="right">25030081</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:191px;">MEM Non-Essential amino acids solution (100X)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">11140050</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Dulbecco&#8217;s Phosphate Buffered saline</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Trypsin-EDTA</td>
			<td style="width:135px;">Gibco</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Fetal bovine serum</td>
			<td style="width:135px;">Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:135px;">EMD Millipore</td>
			<td>MX 1458-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryogenic vial storage boxes</td>
			<td style="width:135px;">Fisherbrand</td>
			<td style="width:128px;">10-500-28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Cryogenic vials</td>
			<td style="width:135px;">Corning/costar</td>
			<td align="right">431416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">T75 flasks</td>
			<td style="width:135px;">Cellstar</td>
			<td align="right" style="width:128px;">658175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">2 well chambered cover glass</td>
			<td style="width:135px;">Nunc</td>
			<td style="width:128px;">155380PK</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Cellometer Vision Cell Profiler</td>
			<td style="width:135px;">Nexcelom Bioscience LLC</td>
			<td style="width:128px;">Cellometer Vision Trio</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Nocodazole</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:128px;">M1404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">CellLight Histone 2B-GFP, BacMam 2.0</td>
			<td style="width:135px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:128px;">C10594</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:191px;">Zeiss Cell Observer Spinning Disk Confocal Microscope system</td>
			<td style="width:135px;">Carl Zeiss Microscopy</td>
			<td style="width:128px;">Zeiss Cell Observer SD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Water Bath</td>
			<td style="width:135px;">Thermoscientific</td>
			<td style="width:128px;">280 series</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Incubator</td>
			<td style="width:135px;">Sanyo commercial solutions</td>
			<td style="width:128px;">MCO-18AIC (UV)</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Class II Biological safety cabinet</td>
			<td style="width:135px;">The Baker Company</td>
			<td style="width:128px;">SterilGard</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Axiovision software</td>
			<td style="width:135px;">Zeiss</td>
			<td style="width:128px;">Ver.4.8.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">ImageJ software</td>
			<td style="width:135px;">National Institute of Health</td>
			<td style="width:128px;">Ver. 1.51r</td>
			<td></td>
		</tr>
	</tbody>
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live cell imaging of chromosome segregation during mitosis

Kromosomer skal være pålideligt og ensartet adskilt i datter celler under mitotiske celledeling. Troskab af kromosomale segregation styres af flere mekanismer, der omfatter spindel forsamling Checkpoint (SAC). SAC er en del af et kompleks feedback-system, der er ansvarlig for forebyggelse af en celle fremskridt gennem mitose, medmindre alle kromosomale kinetochores har vedhæftet en spindel mikrotubuli. Kromosomale halter og unormal kromosom segregation er en indikator for dysfunktionelle cellecyklus kontrol checkpoints og kan bruges til at måle genomisk stabilitet dividere celler. Deregulering af SAC kan resultere i omdannelsen af en normal celle i en maligne celler gennem akkumulering af fejl under kromosomale adskillelse. Gennemførelsen af SAC og dannelsen af den komplekse kinetochore er stramt reguleret af interaktioner mellem kinaser og fosfatase såsom Protein fosfatase 2A (PP2A). Denne protokol beskriver levende celle billeddannelse af halter kromosomer i mus embryonale fibroblaster isoleret fra mus, der havde en knockout af PP2A-B56γ lovgivningsmæssige subunit. Denne metode overvinder manglerne i andre cellecyklus kontrol billeddiagnostiske teknikker såsom flowcytometri eller immuncytokemi, kun giver et øjebliksbillede af en celle cytokinesis status, i stedet for en dynamisk spatiotemporelle visualisering af kromosomer under mitosen.

MEFs fra vildtype og PP2A-B56γ-mus var seedede i en 2-godt kammer dække glas og lov til at vedhæfte. På dag 2, MEFs blev synkroniseret med 0,1% FBS i 24 timer. På dag 3, MEFs i medier med 200 ng/mL nocodazole og 30 PPC CL-HB regentwere inkuberes i 18 timer ved 37° C og 5% CO2. Dag 4, blev cellerne afbildet ved hjælp af en roterende disk Konfokal mikroskop system (figur 1). Live celle imaging blev brugt til at visualisere skæbnen ...

Watch this Scientific Journal Video about Live Cell Imaging of Chromosome Segregation During Mitosis at JoVE.com

Live Cell Imaging of Chromosome Segregation During Mitosis

Denne protokol beskriver en nem og bekvem metode til at mærke og visualisere live kromosomer i mitotiske celler ved hjælp af Histone2B-NGL BacMam 2.0 etiket og et roterende disksystem Konfokal mikroskopi.

Levende celle billeddannelse af kromosom Segregation under mitosen

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

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