Name: viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies
Uploaded: 2015-04-23T14:00:00
Description: Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

Dette arbejde blev støttet af tilskud til JLRR fra: Autisme Taler, Nina Irland, Weston Havens Foundation, NIMH R01 MH081880, og NIMH R37 MH049428. PRW blev støttet af et stipendium fra Science Rådet for Taiwan National. SFS blev støttet af F32 (MH103003).

Etik erklæring: Følgende procedurer er blevet godkendt af vores institution og dyrs protokol. Sørg for at få godkendelse til alle procedurer, der involverer overlevelse operationer, før du begynder eksperimenter og kontrollere alle protokoller er ajour. 1. Lentivirus Forberedelse (valgfrit trin) <ol><li> Split tre 10 cm plader af HEK293T celler for hver lentivirus skal foretages, i nærvær af 10 ml DMEM / 10% FBS, og vokse til ~ 60-70% konfluens. Grow celler i en inkubator v...

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Brugen af GABAerge kortikal interneuron forstadier fra embryonale ganglioniske eminences (GES) for cellebaserede behandlingsformer viser lovende for mange betingelser 12 -1 4. Præcise molekylære teknikker er nødvendige for at spore og hurtig gener af interesse i bestemte interneuron undergrupper. Her giver vi en detaljeret protokol til mærkning embryonale MGE celler med lentivira før transplantationen og vise, hvordan denne teknik kan bruges til at udtrykke gener af interesse i specifikke kortikale inter...

GABAerge corticale interneuroner omfatter ~ 20-30% af neuroner i pattedyr neocortex, mens resten svarer til excitatoriske, glutamaterge vigtigste neuroner. Interneuroner er meget forskellige i elektrofysiologiske egenskaber, axon og dendritceller morfologi og synaptisk målretning 1, og ubalancer i excitatorisk / hæmmende tone er en hypotese at ligge til grund nogle fænotyper af neurologiske / neuropsykiatriske lidelser, herunder autisme, skizofreni og epilepsi 2. Det overordnede mål med protokollen beskrevet heri er at sikre et middel til effektivt at gensplejse GABAerge kortikale interneuron forfædre før transplantation til in vivo analyser. Cortical GABAerge interneuroner er født i den mediale og caudale ganglioniske eminences (MGE og CGE, henholdsvis) 3,4 samt præoptiske område 5. Kortikale interneuron progenitorer undergår langdistance tangential migration fulgtved radial migration for at nå deres endelige mål. Ved ankomsten til deres destinationer, skal disse kortikale interneuroner korrekt integreret i det eksisterende neuronal netværk, og hver unik interneuron undergruppe vil bidrage til kortikale kredsløb på bestemte måder. Fire vigtigste undergrupper kan skelnes ved molekylære markører: MGE-afledt somatostatin (SST) + og parvalbumin (PV) + undergrupper, og CGE-afledt vasoaktivt intestinalt peptid (VIP) + og Reelin +; SST - undergrupper 6. Forskellige corticale interneuron undergrupper fødes over forskellige tidspunkter under fosterudviklingen i MGE og CGE 7, 8. Disse og andre corticale GABAerge interneuron markører er blevet anvendt til at generere specifikke Cre-driver linjer for mange af disse undergrupper 9-11. Transplantation af MGE progenitorer er opstået som en potentiel cellebaseret terapi til behandling af sygdomme, der kan være forårsaget af ubalanceri excitation / hæmning 12 - 24. Disse terapeutiske fordele kan skyldes til dels den unikke evne MGE stamfædre (at sprede, differentiere og integrere i en vært hjernen), eller potentielt fordi mange peri-somatiske hæmmende PV + celler er afledt af MGE. MGE celler kan også hurtigt og effektivt transduceret med lentivirus før transplantation 15 tillader celler, der er genetisk modificeret in vitro til at blive undersøgt in vivo. Begrundelsen for at udvikle denne fremgangsmåde var at overvinde vejspærringer i at studere GABAerge kortikal interneuron udvikling og modning. Især MGE transplantation tilladt forskere til at studere udviklingen af mutante celler in vivo, når mutant mus ellers ville have døde på et tidligt tidspunkt. Ved at indføre gener af interesse før transplantation, virkningerne af specifikke gener på en mutant fænotype kunne vurderes i en efficient måde. Her giver vi en detaljeret protokol til at transducere MGE celler med lentivira før transplantation. Desuden viser vi, hvordan denne teknik kan tilpasses til at udtrykke et gen af ​​interesse i specifikke interneuron undergrupper fra en heterogen gruppe af kortikale interneuron forstadier ved hjælp af en kombination af Cre-afhængige udtryk lentivira og tilgængelige Cre-driver muselinjer. Desuden protokollen introducerer teknikker og en platform for forskere at gensplejse GABAerge kortikal interneuron forstadier til in vivo undersøgelser på en unik måde. En fordel ved denne teknik i forhold til andre aktuelle metoder, er, at de transplanterede MGE celler vil sprede sig væk fra injektionsstedet. Også, i modsætning fokale virale injektioner, efter MGE celler sprede deres morfologi er lettere at vurdere. Denne fremgangsmåde kan anvendes til at undersøge virkningen af ​​indføring af gener af interesse i vildtype- eller mutantceller, indførelse af en celletype specifikreporter at vurdere morfologi eller potentielt at studere virkningen af sygdomstilstande alleler in vivo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and equipment for MGE cell transplantation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>REF 309602</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>301/2 gauge needle</td>
			<td>305106</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>Precision bore to deliver 5 &#181;l (comes with plunger)</td>
			<td>5-000-1005</td>
			<td colspan="2">Drummond scientific company</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td colspan="2">Polysciences, Inc.</td>
		</tr>
		<tr>
			<td>Stereo microscope with boomstand **</td>
			<td>MZ6</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital just for mice stereotaxic instrument **</td>
			<td>51725D</td>
			<td colspan="2">Stoelting company</td>
		</tr>
		<tr>
			<td>Single-axis oil hydraulic fine micromanipulator **</td>
			<td>MO-10</td>
			<td>Narishige</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond coated rotary beveler</td>
			<td>n.a.</td>
			<td colspan="2">made in house</td>
		</tr>
		<tr>
			<td>Needle pipette puller</td>
			<td>730</td>
			<td colspan="2">Kopf instruments</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>n.a.</td>
			<td colspan="2">Any drug store or pharmacy</td>
		</tr>
		<tr>
			<td colspan="2">Any pipette and tips that can reliaby measure 1 &#181;l of volume</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">** These are the particular models we use, but many other setups should work</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Optional Reagents (for making Lentiviruses)</td>
		</tr>
		<tr>
			<td>25 mm, 0.45 &#181;m filter</td>
			<td>09-719B</td>
			<td colspan="2">Fisher Scientific</td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes (25 x 89 mm)</td>
			<td>344058</td>
			<td colspan="2">Beckman Coulter&#174;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (1.5 ml size)</td>
			<td>11668019</td>
			<td colspan="2">Invitrogen</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other commercially available supplies</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid encoding gag/pol</td>
			<td>12251</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev plasmid encoding Rev</td>
			<td>12253</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid encoding VSVG</td>
			<td>12259</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-Flex-GFP</td>
			<td>28304</td>
			<td>Addgene</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

GABAerge kortikale interneuroner, der stammer fra den embryonale mediale og caudale ganglioniske eminences (MGE og CGE), er funktionelt og morfologisk forskelligartet. Indhug er foretaget i forståelsen af ​​roller forskellige kortikale interneuron undergrupper, men der er stadig mange mekanismer, der skal udarbejdes, der kan bidrage til udvikling og modning af forskellige typer GABAerge celler. Endvidere kan ændres GABAergic signalering bidrage til fænotyper af autisme, skizofreni og epilepsi. Specifikke Cre-driver linjer er begyndt at udstykke funktioner unikke interneuron undergrupper. På trods af de fremskridt i musemodeller, er det ofte vanskeligt at effektivt at studere GABAerge kortikale interneuron stamfædre med molekylære metoder in vivo. En vigtig teknik, der anvendes til at studere celleautonom programmering af disse celler er transplantation af MGE celler i værten cortex. Disse transplanterede celler migrere ekstensivt, differentierer etnd funktionelt integrere. Desuden kan MGE celler effektivt transduceret med lentivirus umiddelbart før transplantation, giver mulighed for en lang række molekylære metoder. Her har vi detaljeret en protokol til effektivt at transducere MGE celler før transplantation til in vivo analyse, ved hjælp af tilgængelige Cre-driver linjer og Cre-afhængige ekspressionsvektorer. Denne fremgangsmåde er fordelagtig, fordi den kombinerer præcis genetisk manipulation med evnen af disse celler til at dispergere efter transplantation, hvilket tillader større celletypespecifik opløsning in vivo.

Da MGE celler har den unikke evne til at migrere og integrere når transplanteres i en vært neocortex 16, giver de en fremragende model for genetisk manipulation før in vivo studier. Heri viser vi, hvordan man kan isolere MGE væv fra E13,5 embryoner (figur 1 og 2), som derefter kan transduceres med lentivirus enten in vitro eller i en hurtig måde før transplantation til in vivo undersøgelser. Mærkning MGE via lentivira er udført, før du bruger en forstærke...

Watch this Scientific Journal Video about Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies at JoVE.com

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

GABAerge kortikal interneuron stamfædre sprede, udvikle og synaptisk integrere i et væld cortex efter transplantation. Disse celler kan let transduceres før transplantation til in vivo undersøgelser af genetisk modificerede GABAerge forstadier. Her viser vi virale mærkningsteknikker at målrette specifikke interneuron undergrupper under anvendelse af eksisterende Cre linjer og Cre-afhængige journalister.

Viral-medieret Mærkning og transplantation af Medial ganglioniske Eminence (MGE) Celler til<em&gt; In Vivo</em&gt; Undersøgelser

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

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Developmental Biology

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

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Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Beregningsmæssige analyse af Caenorhabditis elegans kønscelleoverførsel at studere Distribution kerner, proteiner og cytoskelettet

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Encellede transkriptom analyser af mus i bugspytkirtlen endokrine celler

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Intravenøs og Intra-fostervand In Utero Transplantation i Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...