Summary

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em>在体外</em>髓鞘测定

Published: January 12, 2015
doi:

Summary

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.

Abstract

髓鞘是一个复杂的过程,涉及神经元和髓鞘形成神经胶质细胞,在中枢神经系统(CNS)的少突胶质细胞和雪旺氏细胞在周围神经系统(PNS)。我们使用一个体外髓鞘形成测定中,已建立的模型用于研究的CNS髓鞘体外 。要做到这一点,少突胶质细胞前体细胞(OPCs的)被加入到该纯化的初级啮齿类背根神经节(DRG)神经元,以形成髓鞘形成共培养物。为了专门询问角色由少突胶质细胞表达的特定蛋白质发挥对髓鞘形成,我们已经开发协议被接种到DRG神经元在此之前,有选择地使用转导的慢病毒过表达的OPC野生型,组成积极或显性负蛋白质。这让我们专门询问在调节髓鞘少突胶质细胞,这些蛋白质的作用。该协议也可以在加时赛的应用研究她的细胞类型,从而提供了一种方法,允许以期望的细胞类型表达的蛋白质,如少突胶质细胞用于信令和补偿机制的目标研究的选择性操纵。总之,结合慢病毒感染的OPC 体外髓鞘检测提供了参与髓鞘形成的分子机制进行分析的战略工具。

Introduction

轴突的髓鞘是用于在中央和周围神经系统的快速和有效的传输动作电位是至关重要的。特化的细胞,雪旺氏细胞中的外周神经系统和少突胶质细胞在中枢神经系统中,环绕并ensheathe轴突在髓鞘,有效隔热的神经和推动跳跃式传导1。髓鞘形成的方法,可使用的视网膜神经节神经元2中 ,工程化的纳米纤维3,或背根神经节神经元共培养要么雪旺氏细胞4或少突5-7来研究在体外在体外测定髓鞘形成是一个建立的模型用于研究神经系统的髓鞘和它复制许多髓鞘体内 5-8期间发生的基本过程。该法涉及背根神经节(DRG)神经元的纯人口的共培养,用的OPC(对于CNS髓鞘)或雪旺氏细胞(PNS髓鞘)。在特定条件下,这些髓鞘细胞ensheathe DRG轴突在有序,超结构验证,多层状绝缘表达髓鞘本体内特异性蛋白质的相同补质膜片

学习的CNS髓鞘体外的最常用的细胞模型是在共培养物的DRG神经元和OPCs的,并已成功地用于研究的影响,外源因素,如神经营养蛋白产生关于CNS髓鞘体外 5,6。外源因子如生长因子或小分子的药理学抑制剂已被广泛用于研究用DRG-OPC共培养模型7,9的信号通路中髓鞘形成的作用。然而,在包含两个神经元和少突胶质细胞的混合共培养的设置,但仍可能正式,无论是生长因子或所述的医药macological抑制剂可以发挥经DRG神经元和少突胶质细胞(OL)两种效果。这确实提供了具体剖析该蛋白的病种付费仅明示或少突胶质细胞在发挥髓鞘使用这种双电池系统中的作用的能力。明确地确认该信号通路在少突胶质细胞直接调节髓鞘,OPCs的慢病毒转导,对接种到DRG神经元的体外髓鞘测定之前,已被证明是一种优雅的方式来过表达野生型和突变体蛋白质,以及作为组成型表达的蛋白质由少突胶质细胞敲低表达。因此,这种方法提供了一条专门询问和操作信号少突胶质细胞内途径研究髓鞘9,10。

在本文中,我们报告,我们已经发展到过度表达的目的蛋白选择性地少突胶质细胞通过慢病毒的方法方法在体外研究髓鞘形成。该技术始于含有感兴趣的基因的表达载体的产生,无论是在野生型,组成型活性或显性负形式被随后克隆到载体pENTR载体(载体pENTR L1-L2 pENTR4IRES2GFP)。该载体(含有目的基因)中,巨细胞病毒启动子给体(载体pENTR L4-R 1载体pENTR-pDNOR-CMV)和2K7慢病毒结合在酶反应以产生含有CMV启动一个2K7矢量,感兴趣的基因,一个内部核糖体进入位点和GFP( 图1)。该Gateway克隆2K7构建体结合PMD2.G病毒包膜和pBR8.91病毒包可以被共转染到HEK293T细胞,以产生慢病毒随后可以用于转导的OPCs。一旦感染了慢病毒的OPCs的表达感兴趣的蛋白质的高水平。这些OPCs的就可以接种于DRG神经元的文化和效果的表达的高水平的所需蛋白质的施加在髓鞘可以询问。共同评价培养物用免疫印迹分析髓鞘蛋白表达和可视化的免疫细胞化学的形成髓鞘的轴突段。

Protocol

注:用于此研究中的所有动物的混合性别和繁殖的解剖及病理科的动物设施和神经科学与心理健康研究的弗洛里学院在墨尔本大学。所有动物的程序进行,在墨尔本大学经动物实验伦理委员会。 1.克隆2K7慢病毒的前克隆感兴趣的基因插入2K7慢病毒载体,亚克隆的基因导入用标准分子技术11的载体pENTR载体(3637 bp的,卡那霉素抗性)。使用EcoRI和SacⅡ位限制位点?…

Representative Results

旗标记细胞外信号相关 ​​激酶1(标志-ERK1)构建体用于慢病毒的生产用限制性验证酶消化所用的构建体,包括2K7构建体和所需的病毒生产的包装和辅助构建体两者的( 图3) 。 图3:DNA构建验证 。所需的慢病毒生产所有的DNA构建体从Stbl3细菌中纯化,并通过限制?…

Discussion

轴突的髓鞘为脊椎动物的中央和外周神经系统的最佳功能的关键过程。髓轴突的生成和维护是一项涉及神经元之间的分子相互作用,胶质(来自雪旺氏细胞或少突胶质细胞)和细胞外基质蛋白的复杂和协调的过程。这个协议的意义和适用性是它允许操纵蛋白的内混合共培养设置一个特定的细胞类型。作为多种细胞类型中涉及髓鞘在体内和在体外髓鞘测定中,使用钝的药理学工具,以?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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. We would like to acknowledge the Operational Infrastructure Scheme of the Department of Innovation, Industry and Regional Development, Victoria Australia.

Materials

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

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Cite This Article
Peckham, H. M., Ferner, A. H., Giuffrida, L., Murray, S. S., Xiao, J. Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays. J. Vis. Exp. (95), e52179, doi:10.3791/52179 (2015).

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