Name: production and use of lentivirus to selectively transduce primary oligodendrocyte precursor cells for in vitro myelination assays
Uploaded: 2015-01-12T14:00:00
Description: Watch this Scientific Journal Video about Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays at JoVE.com

This work was supported by the Australian National Health and Medical Research Council (NHMRC fellowship #454330 to JX, project grant #628761 to SM and APP1058647 to JX), Multiple Sclerosis Research Australia (MSRA #12070 to JX), the University of Melbourne Research Grant Support Scheme and Melbourne Research CI Fellowship to JX as well as Australia Postgraduate Scholarships to HP and AF.&#160;We would like to acknowledge the Operational Infrastructure Scheme of the Department of Innovation, Industry and Regional Development, Victoria Australia.

BEMÆRK: Alle dyr, der anvendes til denne undersøgelse var af blandet køn og opdrættet på dyrefaciliteter i Anatomisk Institut &amp; patologi og The Florey Institut for Neurovidenskab og Mental Health Research ved University of Melbourne. Alle dyreforsøg blev godkendt af dyreforsøg etiske komitéer på University of Melbourne. 1. Kloning af 2K7 Lentivector <ol><li> Før kloning af genet af interesse i 2K7 lentivirusvektor, subklone genet i pENTR vektoren (3637 bp, kanamycinresistent)...

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Myelinering af axoner er en afgørende proces for den optimale funktion af både det centrale og perifere nervesystem hos hvirveldyr. Frembringelsen og vedligeholdelse af myelinerede axoner er en kompleks og koordineret proces, der involverer molekylære interaktioner mellem neuronal, glial (fra Schwann-celler eller oligodendrocytter) og ekstra cellulære matrixproteiner. Betydningen og anvendelsen af ​​denne protokol er, at det giver mulighed for manipulation af proteiner i en specifik celletype i det blandede co-k...

Myelinering af axoner er afgørende for en hurtig og effektiv overførsel af aktionspotentialer i både det centrale og perifere nervesystem. Specialiserede celler, Schwann-celler i det perifere nervesystem og oligodendrocytter i centralnervesystemet, wrap rundt og ensheathe axoner i myelin, effektivt isolerende nerven og letter saltatory ledning 1. Processen med myelinering kan studeres in vitro ved anvendelse retinale ganglion neuroner 2, manipulerede nanofibre 3 eller dorsale rodganglieneuroner co-dyrket med enten Schwann-celler 4 eller oligodendrocytter 5-7. Den myelinering in vitro-assay er en etableret model til at studere nervesystem myelinering og replikerer mange af de grundlæggende processer, der forekommer under myelinering in vivo 5-8. Assayet involverer cokultur af oprensede populationer af Dorsal rodganglie (DRG) neuroner med OPCs (for CNS myelinering) eller Schwann-celler (for PNS myelinering). Under særlige betingelser disse myelinerende celler ensheathe DRG axoner i bestilt, ultra strukturelt verificeret, multi-lamellar ark af isolerende plasmamembranen, der udtrykker den samme supplement af myelin proteiner til stede in vivo. Den mest almindeligt anvendte celle model studere CNS myelinering in vitro er co-kulturer af DRG-neuroner og OPCs, som med succes er blevet anvendt til at undersøge, at udefrakommende faktorer såsom neurotrophiner udøver på CNS myelinering in vitro 5,6. Eksogene faktorer såsom vækstfaktorer eller småmolekylære farmakologiske inhibitorer er i vid udstrækning blevet anvendt til at undersøge den rolle, signaleringsveje i myelinering anvendelse af DRG-OPC cokultur model 7,9. Men i de blandede co-kultur indstillinger, der indeholder både neuroner og oligodendrocytter, forblev formelt muligt, at enten vækstfaktorer eller Pharmacological hæmmere kunne have udøvet virkninger på både DRG-neuroner og oligodendrocytter (OL). Dette gør tilbyde evnen til specifikt dissekere de roller, de proteiner, der kun udtrykkes af DRG eller oligodendroglia udøver på myelinering ved hjælp af denne dobbelte celle system. Utvetydigt at bekræfte, at signalvejen i oligodendrogliale direkte regulerer myelinering, lentiviral transduktion af OPCs før podning på DRG-neuroner til in vitro myelinering assay, har vist sig at være en elegant måde at overudtrykke både vildtype og mutante proteiner, samt som knockdown ekspression af konstitutivt udtrykte proteiner af oligodendrocytter. Således denne fremgangsmåde giver en avenue til specifikt afhøre og manipulere signalveje inden oligodendrocytter til at studere myelinering 9,10. I dette papir rapporterer vi metoder, som vi har udviklet til at overudtrykke et protein af interesse selektivt i oligodendrocytter via en lentiviralfremgangsmåde til at studere myelinering in vitro. Teknikken begynder med generering af ekspressionsvektorer indeholdende genet af interesse, det være sig i en vildtype, konstitutivt aktiv eller dominant negativ form, der er så efterfølgende klonet ind i pENTR vektoren (pENTR L1-L2 pENTR4IRES2GFP). Denne vektor (indeholdende genet af interesse), er CMV-promoteren donor (pENTR L4-R1 pENTR-pDNOR-CMV) og 2K7 lentivector kombineret i en enzymreaktion til at producere en 2K7 vektor indeholdende CMV promotoren, genet af interesse, en internt ribosomalt indgangssted og GFP (figur 1). Denne gateway klonet 2K7 konstruktion kombineret med PMD2.G viruskappen og pBR8.91 virus pakke kan co-transficeres ind HEK293T celler til at generere lentivirus, der efterfølgende kan anvendes til at transducere OPCs. Når smittet med lentivirus de OPCs udtrykke et højt niveau af proteinet af interesse. Disse OPCs kan derefter podes på DRG neuron kulturer og den virkning, at udtrykketaf høje niveauer af det ønskede protein udøver på myelinering kan aflæses. De co-kulturer vurderes for myelin protein ekspression ved Western blot analyse og visualiseret for dannelsen af ​​myelinerede axonale segmenter ved immunocytokemi.

<table border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Item</td>
			<td style="width:148px;">Manufacturer</td>
			<td style="width:99px;">Catalog #</td>
			<td style="width:183px;">Notes</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">2K7 lentivector&#160;</td>
			<td></td>
			<td></td>
			<td>Kind gift from Dr Suter9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Fluoro-2&#8242;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>F0503-100mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat anti-mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115545205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 goat anti-mouse IgG (H+L)</td>
			<td>Life Technologies</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 goat anti-rabbit IgG (H+L)</td>
			<td>Life Technologies</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A9518-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 - NeuroCul SM1 Neuronal Supplement</td>
			<td>Stem Cell Technologies&#160;</td>
			<td>5711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BDNF (Human)</td>
			<td>Peprotech</td>
			<td>PT450021000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin (d-Biotin)</td>
			<td>Sigma Aldrich</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bradford Reagent</td>
			<td>Sigma Aldrich</td>
			<td>B6916-500ML&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A4161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CNTF</td>
			<td>Peprotech</td>
			<td>450-13020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAKO fluoresence mounting media</td>
			<td>DAKO</td>
			<td>S302380-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, High Glucose, Pyruvate, no Glutamine</td>
			<td>Life Technologies</td>
			<td>10313039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025-375KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS</td>
			<td>Life Technologies</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS, calcium, magnesium</td>
			<td>Life Technologies</td>
			<td>14040182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EBSS</td>
			<td>Life Technologies</td>
			<td>14155063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EcoRI-HF</td>
			<td>NEB</td>
			<td>R3101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Entry vectors for promoter and gene of interest</td>
			<td></td>
			<td></td>
			<td>Generate as per protocols 1-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12003C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forskolin&#160;</td>
			<td>Sigma Aldrich</td>
			<td>F6886-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose (D-glucose)</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Chem Supply</td>
			<td>GL010-500M</td>
			<td>See stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115005003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse IgM&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>115005020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>112005167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Igepal</td>
			<td>Sigma Aldrich</td>
			<td>I3021-100ML&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin&#160;</td>
			<td>Sigma Aldrich</td>
			<td>I6634&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>60615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin&#160;</td>
			<td>Life Technologies</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB Medium</td>
			<td></td>
			<td></td>
			<td>See stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB-Agar</td>
			<td></td>
			<td></td>
			<td>See stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C-7477</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s L-15 Medium</td>
			<td>Life Technologies</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine- 200mM (100X) liquid</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LR Clonase II Plus enzyme</td>
			<td>Life Technologies</td>
			<td>12538-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, NEAA, no Glutamine</td>
			<td>Life Technologies</td>
			<td>10370088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse &#945; &#946;III Tubulin&#160;</td>
			<td>Promega</td>
			<td>G7121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse &#945;MBP (monoclonal)</td>
			<td>Millipore</td>
			<td>MAB381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na pyruvate&#160;</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NAC</td>
			<td>Sigma Aldrich</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NcoI-HF</td>
			<td>NEB</td>
			<td>R3193S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEBuffer 4</td>
			<td>NEB</td>
			<td>B7004S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGF (mouse)</td>
			<td>Alomone Labs</td>
			<td>N-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NT-3</td>
			<td>Peprotech</td>
			<td>&#160;450-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O1 antibody - Mouse anti-O1</td>
			<td>Millipore</td>
			<td>MAB344</td>
			<td>Alternative if O1 hybridoma cells are unavailable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O1 hybridoma cells</td>
			<td></td>
			<td></td>
			<td>Conditioned medium containing anti-O1 antibody to be used for immunopanning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O4 antibody - Mouse anti-O4</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td>Alternative if O4 hybridoma cells are unavailable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O4 hybridoma cells</td>
			<td></td>
			<td></td>
			<td>Conditioned medium containing anti-O4 antibody to be used for immunopanning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Competent Cells</td>
			<td>Life Technologies</td>
			<td>A10460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One Shot Stbl competent cells</td>
			<td>Life Technologies</td>
			<td>C7373-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain Suspension</td>
			<td>Worthington/Cooper</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">pBR8.91</td>
			<td></td>
			<td></td>
			<td>Kind gift from Dr Denham10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDGF-AA (Human)</td>
			<td>Peprotech</td>
			<td>PT10013A500&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin- Streptomycin 100X solution</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pENTRY4IRES2GFP</td>
			<td>Invitrogen</td>
			<td>11818-010&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylenimine (PEI)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-ornithine&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor tablet (Complete mini)</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td></td>
			<td></td>
			<td>Supplied with Clonase enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Putrescine</td>
			<td>Sigma Aldrich</td>
			<td>P-5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit &#945; neurofilament</td>
			<td>Millipore</td>
			<td>AB1987&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit &#945;MBP (polyclonal)</td>
			<td>Millipore</td>
			<td>AB980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ran2 hybridoma cells</td>
			<td>ATCC</td>
			<td>TIB-119</td>
			<td>Conditioned medium containing anti-Ran2 antibody to be used for immunopanning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti CD140A/PDGFRa antibody</td>
			<td>BD Pharmingen</td>
			<td>558774</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SacII</td>
			<td>NEB</td>
			<td>R0157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SOC medium</td>
			<td></td>
			<td></td>
			<td>Supplied with competent bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium selenite&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spe I</td>
			<td>NEB</td>
			<td>R0133S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase Buffer</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer pH8</td>
			<td></td>
			<td></td>
			<td>See stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TNE lysis buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trace Elements B</td>
			<td>Cellgro&#160;</td>
			<td>99-175-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferrin (apo-Transferrin human)</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton&#160;X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T9201-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin Inhibitor From Chicken Egg White</td>
			<td>Roche</td>
			<td>10109878001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (1X), phenol red (0.05%)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Unconjugated Griffonia Simplicifolia Lectin BSL-1</td>
			<td>Vector laboratories&#160;</td>
			<td>L-1100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

production and use of lentivirus to selectively transduce primary oligodendrocyte precursor cells for in vitro myelination assays

Myelinering er en kompleks proces, der involverer både neuroner og myelin danner gliaceller, oligodendrocytter i det centrale nervesystem (CNS) og Schwann-celler i det perifere nervesystem (PNS). Vi anvender en in vitro myelinering assay, en etableret model til undersøgelse CNS myelinering in vitro. For at gøre dette, er oligodendrocyt præcursorceller (OPCs) lægges til den rensede primære gnaver dorsale ganglion (DRG) neuroner til at danne myelinerende co-kulturer. For specifikt at forespørge de roller, som bestemte proteiner udtrykt af oligodendrocytter udøve på myelinering har vi udviklet protokoller, der selektivt transducerer OPCs hjælp af lentivirus overudtrykker vildtype konstitutivt aktive eller dominerende negative proteiner før den podes på DRG-neuroner. Dette giver os mulighed for specifikt at afhøre rollerne for disse oligodendrogliale proteiner i reguleringen myelinering. Protokollerne kan også anvendes i studiet af othendes celletyper, hvilket giver en tilgang, der giver mulighed for selektiv manipulation af proteiner udtrykt af en ønsket celletype, såsom oligodendrocytter for målrettet undersøgelse af signalering og kompensationsordninger. Afslutningsvis kombinere in vitro myelinering assay med lentivirale smittet OPCs giver et strategisk redskab til analyse af molekylære mekanismer involveret i myelinering.

Flag-tagget ekstracellulære signal-relateret kinase 1 (Flag-Erk1) konstruktionen, der anvendes til lentivirus produktion verificeres ved restriktionsenzymspaltning af konstruktionerne anvendes, herunder både 2K7 konstruktioner emballagen og tilbehør konstruktioner kræves til virusproduktion (figur 3) . <img alt="Figur 3" src="/files/ftp_upload/52179/52179fig3highres.jpg" /> Figur 3: DNA-konstruktion kontrol. All...

Watch this Scientific Journal Video about Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays at JoVE.com

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Produktion og anvendelse af Lentivirus til selektivt at transducere Primary oligodendrocyt precursorceller for<em&gt; In vitro</em&gt; myelinering Analyser

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Research

JoVE Journal

Developmental Biology

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Karakterisering af tymisk Bundfældning Progenitorer i Mouse Embryo Brug<em&gt; In vivo</em&gt; Og<em&gt; In vitro</em&gt; Analyser

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro Colony Analyser til karakterisering Tri-potente progenitorceller isoleret fra voksne murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Forberedelse og Kultur Myogenic precursorceller / Primær Myoblaster af skeletmuskulatur af Voksen og Aged Mennesker

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Omprogrammering primære fostervand og membran celler til Pluripotency i Xeno-fri betingelser

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Hurtig påvisning af udviklingsforstyrrelser fænotyper i menneskelige neurale forløber celler (NPC)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Anvendelse af RNAi og Heat-shock-induceret transskription faktor udtryk til at omprogrammere kønsceller til neuroner i C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Single-cell Photoconversion in Living Intact Zebrafish

Encellede Photoconversion i levende intakt zebrafisk

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Spredning og differentiering af Murine myeloide prækursorer 32 d/G-CSF-R celler

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Induktion og validering af cellulære gulne i primære humane celler

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Semi-automatiseret produktion af hepatocyt som celler fra pluripotente stamceller

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...