Name: ex vivo live imaging of single cell divisions in mouse neuroepithelium
Uploaded: 2013-04-30T12:00:00
Description: Watch this Scientific Journal Video about Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium at JoVE.com

Bu araştırma projesi bir ARRA Ek, 5 R01 NS056380 tarafından desteklenmiştir. Ek destek Viral vektör Core ve Emory Nörobilim NINDS Çekirdek Hizmetleri hibe, P30NS055077 en Mikroskopi Çekirdek ile sağlanmıştır. Istikrarlı SSTR3-GFP IMCD3 hücre hattı için Greg Pazour,, Biz GENSAT gelen fare hattı türetmek için Emory Transgenik Fare ve Gen Hedefleme Çekirdek teşekkür ve Sstr3-GFP lentiviral için Bradley Yoder inşa. Monoklonal antikorlar NICHD himayesinde geliştirilen Gelişim Çalışmaları Hibridoma Bankası, elde edilen ve Iowa Üniversitesi, Biyolojik Bilimler Bölümü, Iowa City, IA 52242 tarafından muhafaza edildi. Tüm hayvan prosedürleri IACUC ve Emory Üniversitesi Biyogüvenlik Komitesi tarafından kabul edildi.

Yetişkin fareler, mekanik servikal dislokasyon tarafından ötenazi. Tüm hayvan prosedürleri IACUC ve Emory Üniversitesi Biyogüvenlik Komitesi tarafından kabul edildi. 1. Embriyo Üretimi <ol><li> Çapraz Tamoksifen-indüklenebilir Cre çizgi, CAGGCreER ve dsRedCre muhabiri hattı (Tg (CAG-Bgeo,-DsRed * MST) 1Nagy) (Şekil 1) 5,6. Kızı hücreler, çapraz CAGGCreER ve Olig2-eGFP BAC transgenik farelerin (Tg (Olig2-EGFP) EK23Gsat) (Şekil 1)</stron...

<ol>
	<li>Yamada, T., Pfaff, S. L., Edlund, T., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+pattern+in+the+neural+tube:+motor+neuron+induction+by+diffusible+factors+from+notochord+and+floor+plate.">Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate.</a> Cell. 73, 673-686 (1993).</li><li>Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M., Edlund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+induces+the+differentiation+of+ventral+forebrain+neurons:+A+common+signal+for+ventral+patterning+within+the+neural+tube.">Sonic hedgehog induces the differentiation of ventral forebrain neurons: A common signal for ventral patterning within the neural tube.</a> Cell. 81, 747-756 (1995).</li><li>Briscoe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeodomain+Protein+Code+Specifies+Progenitor+Cell+Identity+and+Neuronal+Fate+in+the+Ventral+Neural+Tube.">Homeodomain Protein Code Specifies Progenitor Cell Identity and Neuronal Fate in the Ventral Neural Tube.</a> Cell. 101, 435-445 (2000).</li><li>Berbari, N. F., Johnson, A. D., Lewis, J. S., Askwith, C. C., Mykytyn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ciliary+localization+sequences+within+the+third+intracellular+loop+of+G+protein-coupled+receptors.">Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors.</a> Mol. Biol. Cell. 19 (4), 1540-1547 (2008).</li><li>Vintersten, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+red:+red+fluorescent+protein+expression+in+mouse+ES+cells,+embryos,+and+adult+animals.">Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.</a> Genesis. 40 (4), 241-246 (2004).</li><li>Hayashi, S., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+recombination+in+diverse+tissue+by+a+tamoxifen-inducible+form+of+Cre:+a+tool+for+temporally+regulated+gene+activation/inactivation+in+the+mouse.">Efficient recombination in diverse tissue by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.</a> Dev. Biol. 244 (2), 305-318 (2002).</li><li>Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., Nowak, N. J., Joyner, A., Leblanc, G., Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425 (6961), 917-925 (2003).</li><li>Mukouyama, Y. S., Deneen, B., Lukaszewicz, A., Novitch, B. G., Wichterle, H., Jessell, T. M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olig2++neuroepithelial+motoneuron+progenitors+are+not+multipotent+stem+cells+in+vivo.">Olig2+ neuroepithelial motoneuron progenitors are not multipotent stem cells in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 103 (5), 1551-1556 (2006).</li><li>Jones, E. A. V., Crotty, D., Kulesa, P. M., Waters, C. W., Baron, M. H., Fraser, S. E., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+in+vivo+imaging+of+postimplantation+mammalian+embryos+using+whole+embryo+culture.">Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture.</a> Genesis. 34, 228-235 (2002).</li><li>Piotrowska-Nitsche, K., Caspary, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+individual+cell+divisions+in+mouse+neuroepithelium+shows+asymmetry+in+cilium+formation+and+Sonic+hedgehog+response.">Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response.</a> Cilia. 1 (6), (2012).</li><li>Nowotschin, S., Ferrer-Vaquer, A., Hadjantonakis, A. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mouse+development+with+confocal+time-lapse+microscopy.">Imaging mouse development with confocal time-lapse microscopy.</a> Methods in Enzymology. 476, 351-377 (2010).</li></ol>

Bizim ex vivo sistem bize doğrudan gerçek zamanlı olarak gelişmekte neuroepithelium içinde tek bir hücre bölünmeleri gözlemlemek sağlar. Örnek olarak fare embriyonik nöral tüp içinde hücre bölünmeleri incelenmiş ve kirpik oluşumu veya Sus yanıt ya izlenir. Biz teknik fizyolojik ilgili verileri sağlar gösteren sabit bölümleri (n = 178) sonuçları ile uyumlu idi bizim görüntüleme sonuçları (n = 24) doğruladı. Bizim teknik tamoksifen do...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J</td>
			<td>Jackson Laboratory</td>
			<td>005438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olig2-eGFP line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd</td>
			<td>MMRRC,</td>
			<td>010555-UCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CAGGCreER</td>
			<td>Jackson Laboratory</td>
			<td>003724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Lonza</td>
			<td>14-416F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat Serum SD male</td>
			<td>Harlan Bioproducts</td>
			<td>4520</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1M Hepes</td>
			<td>BioWhittaker</td>
			<td>17-737E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sstr3-GFP lentivirus</td>
			<td>Emory Viral Core</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro-knife, size 0.025 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>62091</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-L-lysine coated glass bottom dish</td>
			<td>MatTek</td>
			<td>P35GC-0-10-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100% petroleum jelly</td>
			<td>Kroger</td>
			<td>FL9958c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A1R Laser Scanning Confocal Inverted Microscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Imaris 3D software</td>
			<td>Bitplane AG</td>
			<td>Imaris 7.2.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat-inactivated sheep serum</td>
			<td>Invitrogen</td>
			<td>16210-072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafolmaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td>Lab made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-RFP (5F8)</td>
			<td>Chromotek</td>
			<td>110411</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Arl13b serum</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Arl13b 1:5</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Olig2</td>
			<td>Chemicon</td>
			<td>AB9610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Pax6</td>
			<td>Developmental Hybridoma Bank</td>
			<td>Pax6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonalsShh</td>
			<td>Developmental Hybridoma Bank</td>
			<td>5E1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Nkx2.2</td>
			<td>Developmental Hybridoma Bank</td>
			<td>74.5A5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal Ki67</td>
			<td>Abcam</td>
			<td>AB15580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568</td>
			<td>Molecular Probes</td>
			<td>A11031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 350</td>
			<td>Molecular Probes</td>
			<td>A11046</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Fisher</td>
			<td>AC22989</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TO-PRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of single cell divisions in mouse neuroepithelium

Biz floresan işaretleri ve embriyonik fare neuroepithelium kültür dilim canlı görüntüleme entegre bir sistem geliştirdi. Biz izleme soy genetik hücre için mevcut fare hatları yararlandı: Bir Tamoksifen-indüklenebilir Cre hattı ve Cre-aracılı rekombinasyon üzerine dsRed ifade eden bir Cre muhabir hattı. Tamoksifen nispeten düşük düzeyde kullanarak, bize tek tek hücre bölünmeleri takip etmek izin, hücrelerin az sayıda rekombinasyon neden başardık. Ayrıca, bir Olig2-eGFP transgenik çizgi 1-3 kullanarak Sonic Hedgehog (Sus) sinyal için transkripsiyonel yanıt gözlenen ve kirpikler işaretleyici, Sstr3-GFP 4 ifade virüs kültürlü dilim nüfuz ederek kirpikler oluşumu izlenir. Görüntü için neuroepithelium, biz nöral tüp izole, E8.5 embriyolar hasat, görüntüleme odasına uygun kültür koşullarında sinir dilim monte ve zaman atlamalı konfokal görüntüleme yapılır. Bizim eskiin vivo canlı görüntüleme yöntemi bize fizyolojik ilgili bir şekilde birincil kirpikler oluşumu ve Sus cevap göreli zamanlama değerlendirmek için tek bir hücre bölünmeleri izlemek için sağlar. Bu yöntem, kolayca farklı floresan işaretleri kullanarak adapte ve alanında yerinde ve gerçek zamanlı olarak hücre davranışlarını izlemek için hangi ile araçları sağlar olabilir.

Burada E8.5 fare neuroepithelium içinde tek bir hücre bölünmeleri ex vivo canlı görüntüleme yapılır. Tek tek hücrelerin etiketlemek için, rekombinasyon 5,6 (Şekil 3A) üzerine dsRed dile bir Cre muhabir satır içeren bir hücre alt grubunda Cre recombinase bağlı. Böylece, 48 saat sonra biz ex vivo görüntüleme (Şekil 4A-D) sırasında tek bir hücre bölünmeleri gözlemlemek mümkün. 1-3; etiketli hücreleri BAC transgenik Ol...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium at JoVE.com

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Burada için gerekli araçları geliştirmek<em&gt; Ex vivo</em&gt; Canlı görüntüleme fare E8.5 neuroepithelium tek hücre bölünmeleri izlemek için

Ex vivo Fare neuroepithelium Tek Hücre Bölümler Canlı Görüntüleme

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

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JoVE Journal

Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

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Çoklu-fare nöroanatomik Manyetik Rezonans Görüntüleme

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Gelişmekte Neuroepithelium Hücre Davranış Yüksek çözünürlüklü Canlı Görüntüleme

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

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Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

İki-Foton<em&gt; In vivo</em&gt; Bir inceltilmiş-kafatası Hazırlık kullanarak Fare Cortex dendritik dikenler Görüntüleme

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Konfokal Makroskopi kullanarak Postnatal Serebellar Granül Hücre Göç Ex Vivo Görüntüleme

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Eşzamanlı<em&gt; Ex vivo</emİki retina&gt; Fonksiyonel Test tarafından<em&gt; In vivo</em&gt; Elektroretinogram Sistemi

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Tek hücreli çözünürlük Floresans canlı görüntüleme Drosophila sirkadiyen saatler larva beyin kültür

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Ex Vivo Hücre özgü kalsiyum fare diyafram Üçlü sinaps sinyal görüntüleme

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Transpupiller İki Foton Fare Retinasının In vivo Görüntülenmesi

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

In vivo Calcium Imaging in Mouse Inferior Olive

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Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...