从产前Islmn：GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养
1Department of Neurology,Boston Children’s Hospital, 2FM Kirby Neurobiology Center,Boston Children’s Hospital, 3Department of Neurology,Harvard Medical School, 4Medical Genetics Training Program,Harvard Medical School, 5Department of Ophthalmology,Boston Children’s Hospital, 6Department of Ophthalmology,Harvard Medical School, 7Broad Institute of M.I.T. and Harvard, 8Howard Hughes Medical Institute, 9Department of Neurology,Kokura Memorial Hospital, 10Department of Genetics,Albert Einstein College of Medicine
Summary
这项工作提出了一种方案，用于产生原发性耳运动、耳蜗和脊柱运动神经元的同质细胞培养。这些培养物可用于对眼和脊柱运动神经元的形态、细胞、分子和电生理特征进行比较分析。
Abstract
与脊柱运动神经元（SMNs）相比，Oculo运动神经元（CN3s）和血栓神经元（CN4）对退行性运动神经元疾病（如肌萎缩性侧索硬化症（ALS））表现出显著的抵抗力。分离和培养原小鼠CN3、CN4和SM的能力将提供一个研究这种选择性脆弱性背后的机制的方法。迄今为止，大多数协议使用异构细胞培养，这可能混淆实验结果的解释。为了尽量减少与混合细胞种群相关的问题，纯培养物是必不可少的。在这里，第一个协议详细介绍了如何有效地纯化和培养CN3s/CN4s与来自同一胚胎的SMN对应物使用胚胎日11.5（E11.5）Isl MN：GFP转基因小鼠胚胎。该协议提供了关于组织解剖和分离、基于FACS的细胞分离以及CN3/CN4和SMN核细胞体外培养的详细信息。该协议在现有协议中增加了一种新颖的体外CN3/CN4培养系统，同时提供纯物种和年龄匹配的SMN培养物进行比较。以运动神经元的形态、细胞、分子和电生理特性为重点的分析是本培养系统中可行的。该协议将支持研究定义运动神经元发育、选择性脆弱性和疾病的机制。
Introduction
原发性运动神经元的培养是一个强大的工具，它有助于研究神经元发育、功能和对外源应激源的易感性。运动神经元培养对神经退行性疾病的研究特别有用，如肌萎缩性侧索硬化症（ALS）1，2，其疾病机制尚未完全了解。有趣的是，尽管ALS患者和ALS模型小鼠的脊髓运动神经元（SMNs）的细胞死亡显著，但神经运动神经元（CN3s）和脑神经（CN4s）中的细胞死亡相对稀少，1、3、4、5、6、7、8、9。因此，对CN3s/CN4s和SMN的纯文化进行比较分析，可以为相对脆弱性背后的机制提供重要的线索。不幸的是，这种分析的一个主要障碍是无法生长这些运动神经元的纯化培养物。
许多协议已经描述从动物模型净化SBN。这些协议大多使用密度梯度离心10，11，12和/或p75NTR-抗体基细胞分选平移技术13，14，15，16。密度梯度离心利用相对于其他脊髓细胞更大的SMN，而p75NTR是脊髓中SMN专门表达的细胞外蛋白。近100%纯SMN文化是由这些协议11、12、14的一个或两个产生的。然而，这些协议在生成CN3/CN4培养物方面尚未成功，因为CN3s/CN4s没有表达p75NTR，其他特定的CN3/CN4标记尚未识别。它们也比 SMN 小，因此，根据大小更难隔离。相反，CN3s或CN4的体外研究依赖于分离的17，18，19，20，21，除植物17，22，23，24，25，26和切片27，28培养基，这些培养体由异质细胞类型组成，并且不存在任何协议。原发性CN3或CN4的隔离和培养。
在这里，描述了一个协议，用于从同一胚胎日11.5 （E11.5） IslMN：GFP转基因小鼠29（图1，图2A）对CN3、CN4s和SM的可视化、隔离、纯化和培养。IslMN：GFP特别标记运动神经元与远鼻化 GFP，本地化到细胞膜。该协议使多种运动神经元的物种和年龄匹配比较，以阐明运动神经元疾病的病理机制。
Protocol
所有利用实验室动物的实验都是按照NIH关于照顾和使用实验室动物的准则进行的，并经波士顿儿童医院动物护理和使用委员会批准。 1. 在解剖前设置定时配合 为了产生用于运动神经元收获的产前胚胎小鼠，在神经元分离前11.5天对每只雌性小鼠进行称重，并在成年IslMN：GFP转基因小鼠之间建立定时交配。为了开发该协议，使用129S1/C57BL/6J IslMN：GFP</…
Representative Results
该协议的目的是高度净化和培养初级CN3s/CN4s和SMN长期，以便能够比较分析运动神经元紊乱背后的机制（见图1和图2概述）。 一旦神经元在培养中成功分离和生长，几乎纯原发的CN3/CN4和SMN培养物获得（图5A，B），并维持至少14DIV（图4…
Discussion
从历史上看，CN3和/或CN4运动神经元的体外研究依赖于异质培养物，如分离17、18、19、20、21、外植17、22、23、24、25、26和<sup …
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢布里吉特·佩特曼（生物根，剑桥，马萨诸塞州，美国）在SMN解剖技术的指导;达纳法伯癌症研究所流式细胞测定设施、哈佛医学院免疫学部门流式细胞测定设施、乔斯林糖尿病中心流式细胞测定中心、布里格姆和妇女医院流动细胞学核心，以及波士顿儿童医院流式细胞测定研究设施，用于FACS对原发性运动神经元进行FACS分离;A.A. Nugent、A.P.Tenney、A.S.Lee、E.H.Nguyen、M.F.Rose、其他恩格尔实验室成员和项目ALS联盟成员提供技术援助和深思熟虑的讨论。这项研究得到了ALS项目的支持。此外，R.F.还由日本心脏基金会/拜耳雅库因海外研究赠款和NIH遗传学T32 GM007748培训赠款资助;J.J.通过舍彭斯眼科研究所和开发神经学训练计划博士后奖学金（5T32NS0007473-19）通过波士顿儿童医院，获得美国国家卫生研究院/NEI眼病分子库（5T32EY00714-16）培训项目的支持;M.C.W得到了NEI（5K08EY027850）和儿童医院眼科基金会（教师发现奖）的支持;和E.C.E.是霍华德·休斯医学研究所的调查员
Materials
| Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) | Thermo Fisher Scientific | A-11001 | 1:400 |
| Alexa Fluor 594-conjugated F(ab')2 goat anti-rabbit IgG (H+L) | Thermo Fisher Scientific | A-11072 | 1:400 |
| B27 Supplement (50X), serum free | Thermo Fisher Scientific | 17504-044 | |
| BD FACSAria llu SORP Flow Cytometer | BD Bioscience | – | This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers. |
| BD Falcon 70μm Nylon Cell Strainers | CORNING | 352350 | For filtering the dissociating cells before FACS. |
| BD Falcon Round Bottom Test Tubes With Snap Cap | CORNING | 352054 | |
| BDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-207 | |
| Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well) | Greiner Bio-One International | 655090 | We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC |
| Circular Cover Glasses for microscopy | Karl Hecht & Assistent | 1001/14 | We used this coverslip since the area was large (diamater: 14 mm). |
| CNTF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-272 | |
| Cyclopiazonic acid from Penicillium cyclopium | Sigma-Aldrich | C1530 | CPA. One of ER stressors. |
| 4′,6-diamidino-2-phenylinodole (DAPI) | Thermo Fisher Scientific | D1306 | |
| Dimethyl sulfoxide | Sigma-Aldrich | D2650 | DMSO |
| Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips | Roboz Surgical Instrument | RS-5015 | |
| Forskolin | Thermo Fisher Scientific | BP25205 | |
| GDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-305 | |
| GlutaMAX supplement | Thermo Fisher Scientific | 35050-061 | |
| Hanks’ Balanced Salt Solution (HBSS) | Thermo Fisher Scientific | 14175-095 | |
| Hibernate E | BrainBits | HE | |
| Hibernate E low fluorescence | BrainBits | HELF | Fluorescence which hinders observation of embryo's GFP expressions should be low. |
| Horse serum, heat inactivated, New Zealand origin | Thermo Fisher Scientific | 26050-070 | |
| IBMX | Tocris Cookson | 2845 | Isobutylmethylxanthine |
| Laminin | Thermo Fisher Scientific | 23017-015 | |
| Leibovitz’s L15 medium | Thermo Fisher Scientific | 11415064 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| Micro Dissecting Scissors | Roboz Surgical Instrument | RS-5913 | |
| Micro Knife 4.75" 1.7 x 27 mm blade | Roboz Surgical Instrument | RS-6272 | |
| Moria Mini Perforated Spoon | Fine Science Tools | 10370-19 | |
| mouse monoclonal antibody to neuronal class III β-tubulin (TUBB3) | BioLegend | 801202 | 1:500, TUJ1 |
| Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements software | Nikon | – | Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments |
| Nitric Acid 90%, Fuming (Certified ACS) | Fisher Scientific | A202-212 | For rinsing coverslips |
| Olympus 1.7ml Microtubes, Clear | Genesee Scientific | 22-281 | These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context. |
| Papain Dissociation System | Worthington Biochemical Corp | LK003150 | Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit. |
| Penicillin-streptomycin (10,000 U/ml) | Thermo Fisher Scientific | 15140-122 | |
| Phosphate buffered saline (PBS) | Thermo Fisher Scientific | 10010-023 | |
| Poly D-lysin (PDL) | MilliporeSigma | A-003-E | |
| rabbit monoclonal antibody to Islet1 | Abcam | ab109517 | 1:200 |
| SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera | Nikon | – | Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes. |
| Sylgard 170 Black Silicone Encapsulant – A+B 0.9 Kg kit | Dow Corning | 1696157 | We make dissection dishes using this kit. |
| TC treated Dishes, 100 x 20 mm | Genesee Scientific | 25-202 | We make dissection dishes using this dish. |
| Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip Width | Roboz Surgical Instrument | RS-8100 | |
| Transducer for LOGOQ e VET | GE Healthcare | L8-18i-RS | For ultrasound on female mice |
| Veterinary ultrasound machine | GE Healthcare | LOGOQ e VET | For ultrasound on female mice |
| Zeiss LSM 700 series laser scanning confocal microscope and Zen Software | Carl Zeiss | – | Confocal image of the embryo was captured with these equipments |
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Cite This Article
Fujiki, R., Lee, J. Y., Jurgens, J. A., Whitman, M. C., Engle, E. C. Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice. J. Vis. Exp. (153), e60440, doi:10.3791/60440 (2019).