在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素
Summary
我们提出了一个协议,使用前向遗传方法筛选在果蝇黑色素细胞中表现出神经退化的突变体。它结合了爬升测定、组织分析、基因映射和DNA测序,最终鉴定出与神经保护过程相关的新基因。
Abstract
关于神经退行性疾病的发病和进展,包括负责的基本基因,有许多需要了解。使用化学诱变剂进行前向基因筛选是一种有用的策略,用于将突变表型与果蝇和其他与人类共享保守细胞途径的模型生物体之间的基因进行映射。如果感兴趣的突变基因在苍蝇的早期发育阶段不是致命的,则可以进行爬升测定,以筛选大脑功能下降的样型指标,如低爬升率。随后,对脑组织进行二次组织学分析,通过评分神经变性表型来验证基因的神经保护功能。基因映射策略包括依赖这些相同测定的微量和缺陷映射,可以遵循DNA测序来识别感兴趣的基因中可能存在的核苷酸变化。
Introduction
神经元在大部分后是线粒体,不能分裂1,2。在大多数动物中,神经保护机制的存在,以维持这些细胞在整个生物体的整个寿命,特别是在老年时,神经元最容易受到损害。这些机制背后的基因可以在出现神经退化的突变体中识别,这是一种表型指标,用于神经保护的丧失,使用正向遗传方案。使用化学诱变剂(如乙酸甲酸酯 (EMS) 或 N-乙基-N-硝基苏雷亚 (ENU) 的正向基因筛网特别有用,因为它们诱发随机点突变,从而产生一种固有的不偏不倚的方法,揭示了真核模型生物体中的大量基因功能(相反,X射线诱变会产生DNA断裂,并可能导致重排而不是点突变6)。
常见的果蝇果蝇黑色素是这些屏幕的理想主题,因为它的高品质,有编号的基因组序列,其长期历史作为一个模型生物与高度发达的遗传工具,最重要的是,其共享进化史与人类7,8。该协议的适用性的一个限制因素是由突变的基因引起的早期杀伤力,这将阻止在9岁时的测试。然而,对于非致命突变,利用负地轴的爬升测定是一种简单的,虽然广泛的,量化受损的运动功能10的方法。为了表现出足够的运动反应性,苍蝇依靠神经功能来确定方向、感知其位置和协调运动。因此,苍蝇无法充分攀爬以响应刺激,这可能表明神经缺陷11。一旦发现特定的有缺陷的攀爬表型,使用辅助屏幕(如脑组织分析)进行进一步测试,可用于识别攀爬缺陷苍蝇的神经退化。随后的基因映射可用于揭示染色体上的基因组区域,该染色体携带感兴趣的有缺陷的神经保护基因。为了缩小感兴趣的染色体区域,可以使用携带具有染色体上已知位置的显性标记基因的突变飞线进行中度映射。标记基因作为突变的参考点,因为两个位点之间的重组频率提供了一个可测量的距离,可用于绘制基因的大致位置。最后,在感兴趣的染色体色谱区域上,用带有平衡缺陷的线穿过突变线,创建一个补充测试,其中,如果感兴趣的基因表示其已知的表型5,则可以验证该基因。识别基因中的多态核苷酸序列,可能导致氨基酸序列的改变,可以通过测序该基因并将其与果蝇基因组序列进行比较来评估。后续感兴趣的基因表征可以包括测试额外的突变等位基因,突变抢救实验和检查额外的表型。
Protocol
1. 苍蝇的准备和老化 获取或生成6个将被用于基因筛选的果蝇突变体的集合。在这里,使用 ENU 诱变线映射到第二个染色体,并在CyO上平衡。 在25°C、12小时光/暗循环的培养箱中放大实验基因型系,在玉米粉-糖蜜培养基上。 从每个实验基因型中收集大约20个同源后代,在0-2天的成人环状之间。通过一贯收集雄性或雌性苍蝇来消除性别偏见。 ?…
Representative Results
在此分析中,我们介绍了用于识别基因脑肿瘤(brat)的步骤,这些步骤在维持成年苍蝇17的神经元完整性(例如神经保护)方面起着作用;一种可用于识别神经保护中涉及的基因的方法。我们使用一种正向遗传方法(该策略如图1A中概述)使用爬升测定筛选化学诱变苍蝇的集合(用于此测定的仪器如图1B所示)。?…
Discussion
果蝇的向前基因筛选是识别不同生物过程所涉及的基因的有效方法,包括年龄依赖性神经保护5,23,24,25.使用这种策略,我们成功地鉴定了布拉特为一种新的神经保护基因17。
该协议的一个关键步骤涉及组织学分析的头部的正确方向(如第 4.4.3 节所述)。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们特别感谢巴里·加内茨基博士,他在实验室里进行了基因筛选,允许识别和鉴定小布作为神经保护基因。我们感谢史蒂文·罗比诺博士提供本文中介绍的基因屏幕中使用的ENU诱变苍蝇的集合。我们感谢Ganetzky实验室的成员,格雷斯·博克霍夫-福尔克博士和大卫·瓦萨曼博士在整个项目期间进行有益的讨论,何玲玲和鲍勃·克雷伯提供了技术援助,池田Aki博士在威斯康星大学和金·莱基博士和阿拉巴马大学光学分析设施。
Materials
| Major equipment | |||
| Fume hood for histology | |||
| Light Microscope | Nikon | Eclipe E100 | Preferred objective for imaging is X20 |
| Imaging software | Nikon | ||
| Microscope Camera | Nikon | ||
| Thermal cycler | Eppendorf | ||
| Fly pushing and climbing assay | |||
| VWR® Drosophila Vial, Narrow | VWR | 75813-160 | |
| VWR® General-Purpose Laboratory Labeling Tape | VWR | 89097-912 | |
| Standard mouse pad | |||
| Stereoscope | Motic | Model SMZ-168 | |
| CO2 anesthesia station (Blowgun, foot valve, Ultimate Flypad) | Genesee Scientific | 54-104, 59-121, 59-172 | Doesn’t iinclude CO2 tank |
| Fine-Tip Brushes | SOLO HORTON BRUSHES, INC. | ||
| Drosophila Incubator | VWR | 89510-750 | |
| Gene mapping | |||
| CantonS | Bloomington Drosophila Stock Center | 9517 | |
| w1118 | Bloomington Drosophila Stock Center | 5905 | |
| yw | Bloomington Drosophila Stock Center | 6599 | |
| Drosophila line used for recombination mapping | Bloomington Drosophila Stock Center | 3227 | Genotype: wg[Sp-1] J[1] L[2] Pin[1]/CyO, P{ry[+t7.2]=ftz/lacB}E3 |
| CyO/sno[Sco] | Bloomington Drosophila Stock Center | 2555 | Drosophila balancer line used for recombination mapping |
| Deficiency Kit for chromosome 2L | Bloomington Drosophila Stock Center | DK2L | Cook et al., 2012 |
| Histology analysis | |||
| Ethanol, (100%) | Thermo Fischer Scientific | A4094 | |
| Chloroform | Thermo Fischer Scientific | C298-500 | |
| Glacial Acetic Acid | Thermo Fischer Scientific | A38-500 | |
| Fisherbrand™ Premium Microcentrifuge Tubes: 1.5mL | Thermo Fischer Scientific | 05-408-129 | |
| Histochoice clearing agent 1X | VWR Life Sciences | 97060-934 | |
| Harris Hematoxylin | VWR | 95057-858 | |
| Eosin | VWR | 95057-848 | |
| Thermo Scientific™ Richard-Allan Scientific™ Mounting Medium | Thermo Scientific™ 4112 | 22-110-610 | CyO/sna[Sco] |
| Unifrost Poly-L-Lysine microscope slides, 75x25x1mm, EverMark Select Plus | Azer Scientific | ||
| Fisherbrand™ Cover Glasses: Rectangles | Fisherbrand | 12-545M | Dimensions: 24×60 mm |
| Traceable timer | VWR | ||
| Slide Warmer | Barnstead International | model no. 26025 | |
| Slide tray and racks | DWK Life Sciences | Rack to hold 20 slides | |
| Fisherbrand™ General-Purpose Extra-Long Forceps | Fisherbrand | 10-316A | |
| Kimwipes™ | Kimberly-Clark™ Professional | ||
| 6 inch Puritan applicators | Hardwood Products Company, Guilford, Maine | 807-12 | |
| VWR® Razor Blades | VWR | 55411-050 | |
| Tupperware or glass containers for histology liquids | 16 + 1 for running water | ||
| High Profile Coated Microtome Blades | VWR | 95057-834 | |
| Corning™ Round Ice Bucket with Lid, 4L | Corning™ | ||
| Beaker | Or other container for ice water and cassettes | ||
| Tissue Bath | Precision Scientific Company | 66630 | |
| Microtome | Leica Biosystems | ||
| Molecular analysis | |||
| Wizard® SV Gel and PCR Clean-Up System | Promega | A9282 | |
| Ex Taq DNA polymerase | TaKaRa | 5 U/μl | |
| Invitrogen™ SYBR™ Safe™ DNA Gel Stain | Invitrogen™ | ||
| UltraPure™ Agarose | Invitrogen™ | ||
| 1 Kb Plus DNA Ladder | Invitrogen™ | ||
| ApE-A plasmid Editor software | Available for free download | ||
| Statistical analysis | |||
| R software package | |||
| Further analysis | |||
| y[1] w[*]; wg[Sp-1]/CyO; Dr[1]/TM3, Sb[1] | Bloomington Drosophila Stock Center | 59967 | |
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Citazione di questo articolo
Gevedon, O., Bolus, H., Lye, S. H., Schmitz, K., Fuentes-González, J., Hatchell, K., Bley, L., Pienaar, J., Loewen, C., Chtarbanova, S. In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster. J. Vis. Exp. (149), e59720, doi:10.3791/59720 (2019).