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

Ce projet de recherche a été soutenue par un supplément ARRA, 5 R01 NS056380. Un soutien supplémentaire a été fourni par le vecteur viral de base et le noyau de microscopie de l&#39;Emory Neuroscience NINDS les principales installations de subvention, P30NS055077. Nous remercions la souris transgénique Emory et Gene Targeting base pour dériver la lignée de souris à partir de GENSAT; Greg Pazour pour la lignée cellulaire SSTR3-GFP IMCD3 stable et Bradley Yoder pour le SSTR3-GFP lentiviral construisons. Les anticorps monoclonaux ont été obtenus à partir des études du développement Hybridoma Bank, élaboré sous les auspices du NICHD, et entretenus par l&#39;Université de l&#39;Iowa, Département des sciences biologiques, Iowa City, IA 52242. Toutes les procédures d&#39;animaux ont été approuvés par le IACUC et le Comité de biosécurité de l&#39;Université Emory.

souris adultes sont euthanasiés par dislocation cervicale mécanique. Toutes les procédures d&#39;animaux ont été approuvés par le IACUC et le Comité de biosécurité de l&#39;Université Emory. 1. Embryon Generation <ol><li> Cross tamoxifène Cre inductible ligne, CAGGCreER, et la ligne de journaliste dsRedCre (Tg (CAG-Bgeo,-DsRed * MST) 1Nagy) (Figure 1) 5,6. Pour suivre la chronologie relative de la réponse Shh dans les cellules filles, croix CAGGCreER...

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Notre système ex vivo nous permet d&#39;observer directement divisions cellulaires uniques dans le neuroépithélium développement en temps réel. À titre d&#39;exemple, nous avons examiné les divisions cellulaires dans le tube neural embryonnaire de la souris et surveillés soit la formation cil ou réponse Shh. Nous avons confirmé nos résultats d&#39;imagerie (n = 24) ont été cohérents avec les résultats des sections fixes (n = 178) indiquant notre technique fournit des données ph...

<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

Nous avons développé un système qui intègre l&#39;imagerie en direct de marqueurs fluorescents et des tranches de culture du neuro-épithélium embryonnaire de la souris. Nous avons profité de lignées de souris existants pour la cellule génétique lignée de traçage: une ligne Cre inductible par le tamoxifène et une ligne de journaliste exprimant Cre DsRed lors de la recombinaison Cre-médiation. En utilisant un niveau relativement bas du tamoxifène, nous étions capables d&#39;induire la recombinaison dans un petit nombre de cellules, ce qui permet de suivre les divisions cellulaires individuels. En outre, nous avons observé la réponse transcriptionnelle à Sonic Hedgehog (Shh) de signalisation utilisant un Olig2-eGFP transgénique ligne 1-3 et nous avons suivi la formation des cils en infectant la tranche cultivé avec le virus exprimant le marqueur de cils, SSTR3-GFP 4. Afin d&#39;image à l&#39;neuroépithélium, nous avons récolté embryons à E8.5, isolé du tube neural, fixé la tranche de neurones dans des conditions de culture appropriées dans la chambre de formation d&#39;image et réalisée en accéléré imagerie confocale. Notre exvivo méthode imagerie en temps réel permet de tracer des divisions cellulaires simples pour évaluer la chronologie relative de la formation des cils primaires et la réponse Shh d&#39;une manière physiologiquement pertinents. Cette méthode peut être facilement adapté en utilisant des marqueurs fluorescents distinctes et fournit le terrain, les outils permettant de surveiller le comportement des cellules in situ et en temps réel.

Ici, nous avons effectué ex vivo l&#39;imagerie en direct de divisions cellulaires simples dans le neuroépithélium souris E8.5. Pour marquer les cellules individuelles, nous avons provoqué la recombinase Cre dans un sous-ensemble de cellules contenant une ligne de journaliste Cre celle exprimée DsRed lors de la recombinaison 5,6 (figure 3A). Ainsi, 48 heures plus tard, nous avons pu observer des divisions cellulaires simples pendant ex imagerie in vivo ...

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

Ici, nous développons les outils nécessaires pour<em&gt; Ex vivo</em&gt; Imagerie en temps réel à tracer des divisions cellulaires simples dans la souris E8.5 neuroépithélium

Ex vivo d&#39;imagerie en temps réel de simples divisions cellulaires dans neuroépithélium de souris

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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Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

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