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

Dit onderzoek werd ondersteund door een ARRA Supplement, 5 R01 NS056380. Extra steun werd verleend via de virale vector Core en de Microscopie Kern van de Emory Neuroscience NINDS Core Facilities subsidie, P30NS055077. Wij danken de Emory Transgene muis en gene targeting Core voor het afleiden van de muis lijn van GENSAT; Greg Pazour voor de stabiele SSTR3-GFP IMCD3 cellijn, en Bradley Yoder voor de Sstr3-GFP lentivirale construct. Monoklonale antilichamen werden verkregen van de Developmental Studies Hybridoma Bank, ontwikkeld onder auspiciën van de NICHD, en onderhouden door de Universiteit van Iowa, Afdeling Biologische Wetenschappen, Iowa City, IA 52242. Alle dierlijke procedures werden goedgekeurd door de IACUC en het Biosafety Comite aan de Emory University.

Volwassen muizen geëuthanaseerd mechanisch cervicale dislocatie. Alle dierlijke procedures werden goedgekeurd door de IACUC en het Biosafety Comite aan de Emory University. 1. Embryo Generation <ol><li> Cross tamoxifen-induceerbare Cre lijn, CAGGCreER en dsRedCre reporter lijn (Tg (CAG-Bgeo,-DsRed * MST) 1Nagy) (Figuur 1) 5,6. Voor het bewaken van de relatieve timing van Shh respons in de dochtercellen, kruis CAGGCreER en DsRed lijn met de Olig2-eGFP BAC transg...

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Onze ex vivo systeem stelt ons in staat om direct waar te nemen enkele cel verdeeldheid binnen de ontwikkeling neuroepithelium in real time. Als voorbeeld nemen we onderzocht celdelingen in de muis embryonale neurale buis en bewaakt ofwel cilium vorming of Shh respons. We bevestigden onze beeldvorming resultaten (n = 24) waren in overeenstemming met de resultaten uit vaste rubrieken (n = 178) met vermelding van onze techniek biedt fysiologisch relevante gegevens. D...

<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

We ontwikkelden een systeem dat levende beeldvorming van fluorescente markers en het kweken plakjes embryonale muis neuroepithelium integreert. We hebben gebruik gemaakt van de bestaande muis lijnen voor genetische cell lineage tracing: een tamoxifen-induceerbare Cre lijn en een Cre reporter lijn expressie DsRed bij Cre-gemedieerde recombinatie. Door het gebruik van een relatief laag niveau van tamoxifen, waren we in staat om recombinatie te induceren in een klein aantal cellen, waardoor ons toe om individuele divisies cel volgen. Daarnaast zagen we de transcriptionele respons op Sonic Hedgehog (Shh) signalering met behulp van een Olig2-eGFP transgene lijn 1-3 en we gecontroleerd vorming van cilia door de gekweekte slice met virus dat de trilharen marker, Sstr3-GFP 4 infecteren. Om het imago van de neuro-epitheel, we geoogst embryo&#39;s in E8.5, geïsoleerd van de neurale buis, bevestigd de neurale schijfje in de juiste kweekomstandigheden in de beeldvorming kamer en deed time-lapse confocale beeldvorming. Onze exvivo live-imaging methode stelt ons in staat om enkele celdelingen traceren om de relatieve timing van primaire cilia vorming en Shh reactie in een fysiologisch relevante wijze kan worden gemeten. Deze methode kan eenvoudig worden aangepast met behulp van verschillende fluorescente markers en geeft het veld de instrumenten waarmee naar cel gedrag in situ en in real time te volgen.

Hier voerden we ex vivo live-beeldvorming van enkele cel verdeeldheid binnen de E8.5 muis neuroepithelium. Om individuele cellen te labelen, we geïnduceerde Cre recombinase in een subset van cellen met een Cre reporter lijn die uitgedrukt DsRed na recombinatie 5,6 (figuur 3A). Zo, 48 uur later waren we in staat om enkele celdelingen tijdens ex vivo beeldvorming (Figuren 4A-D) te observeren. Gelijktijdig, we gecontroleerd wanneer de gelabelde cellen werd Shh...

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

Hier ontwikkelen we de instrumenten die nodig zijn voor de<em&gt; Ex vivo</em&gt; Live-imaging tot enkele celdelingen in de muis E8.5 neuroepithelium traceren

Ex vivo Live-Imaging van Single Cell Divisies in Muis neuroepithelium

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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Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

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