正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选
Summary
应力性的标志长寿之一，已知是基因支配。在这里，我们开发了一个不带偏见的高通量方法筛选，赋予抗逆性ES细胞以开发小鼠模型长寿研究突变。
Abstract
Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.
Introduction
长寿与抗逆性的亲密关系。在一般情况下，长寿命的物种通常显示增加的阻力向着多重压力，例如过氧化氢 ，百草枯（PQ），紫外线，热，和重金属1,2。与此相反，对压力增加的灵敏度趋向于预测寿命缩短和/或更多的疾病倾向的表型。抗氧化剂的清除途径早已推测在赋予抗逆性的动物中发挥重要作用。然而，除少数例外，从各种转基因动物的与各种抗氧化剂的酶（例如，超氧化物歧化酶）的操作的研究表明，增加的氧化剂清除酶的水平没有增加的寿命或健康跨度3。这些数据表明，在应力抗性性状在长寿命的动物始终观察由其他细胞途径尚未被揭露介导的。
我们采取了一个不带偏见向前遗传的方式来确定的基因，其在突变，可以赋予胁迫抗性表型中培养的胚胎干细胞（ES）细胞。 ES细胞提供在这项研究中两个主要的优点：（1）复杂的遗传操作可用于修改ES细胞的基因组中;和（2）的任何应力耐性ES细胞从屏幕中回收可直接用于鼠标的生产，从而允许快速翻译成整个动物研究来测量寿命和健康跨度。
在本报告中，我们描述了使用C9胚胎干细胞系，其中脂双层等位基因控制下由一个四环素响应元件。多西环素（DOX）治疗短暂关闭，博莱霉素导致姐妹染色单体交换的增加事件的表达。这种短期博来霉素敲除允许的杂人口内纯合突变的产生，使隐性突变为抗逆性可在筛选过程中被捕获。我们也描述了使用座子piggyBac（PB）作为转座子诱变随机插入 聚-A捕获磁带（PB-UPA）变异基因在基因组中。细胞与基因由聚甲陷阱的破坏成为G418抗性和可被回收，这样的基因陷阱突变（基因陷阱库）的集合可以作出，并随后筛选突变体克隆是耐应力。
应力从选择回收的抗性克隆可以被相当迅速特征在于分子技术中插入的数目（定量PCR）的方面，插入部位（splinkerette PCR），被破坏的基因（BLAST）的身份，并且其表达水平（ RT-qPCR的）。 该 PB插入可以通过MPB转座的瞬时表达在克隆被重新活化用来恢复野生型DNA序列，从而测试应力阻力的损失。这些是有效的方法来确认突变的因果关系，应事先做昂贵的鼠标生产。以往的研究表明，细胞暴露在压力失去了他们的多能性4,5。因此，在该协议中，一个副本集突变的细胞，这将不能与压力处理的保存是成功小鼠生产的关键。
我们的实验室已经设计了C9胚胎干细胞系和PB-UPA载体，两者都可以应要求其他调查。这里所报告的协议将由从头文库基因束缚ES细胞用PB-UPA（图1A）中，然后影印和应力选以分离应力抗性克隆（ 图1B）的产生开始。我们证明百草枯，在细胞内一种强力游离自由基产生剂的选择。实际上，任何细胞毒性化合物或毒素，例如，ER 压力 （如，毒胡萝卜素和衣霉素），神经元氧化剂（例如，MPP +处理，6-羟基多巴胺，和鱼藤酮），热 ，和重金属 （如，镉，硒），可以适用于该方法以选择用于各个抗性突变体。
Protocol
Representative Results
Discussion
Forward genetic analysis allows for an unbiased interrogation of the genome for genes responsible for a specific phenotype. This method is very powerful to uncover novel gene functions. It has been widely used in lower organisms but not in mammal, such as the mouse, mainly due to the extremely high cost associated with the infrastructure and logistics that would entail. Here, we moved the genetic screening process to the ES cell culture platform, greatly increasing the efficiency and throughput in generating mutants a…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank the Wellcome Trust Sanger Institute for the gifts of piggyBac transposon and piggyBac transposase. This work was supported by the Butcher grant of Colorado and the NIH R01 AG041801 (W.S.C).
Materials
| Vector | ||
| PB-UPA | ||
| mPBase | ||
| mPBasePuro | ||
| Tissue Culture | ||
| 500-ml Stericup filters | EMD Millipore | SCGPU05RE |
| 250-ml Stericup filters | EMD Millipore | SCGPU02RE |
| 50-ml Steriflip-GV filters | EMD Millipore | SE1M179M6 |
| KO DMEM | Life Technologies | 10829-018 |
| DMEM | Sigma-Aldrich | D6429 |
| FBS | Tissue Culture Biologicals | 104 |
| Heat Inactivated FBS | Sigma-Aldrich | F4135-500 |
| LIF | EMD Millipore | ESG1107 |
| Non-essential Amino Acids | Life Technologies | 11140-050 |
| GlutaMAX | Life Technologies | 35050-061 |
| Pen/Strep | Life Technologies | 15140148 |
| β-Mercaptoethanol | Life Technologies | 21985-023 |
| Methyl Viologen dichloride (Paraquat) | Sigma-Aldrich | 856177 |
| Dimethyl Sulphoxide Hybri-MAX | Sigma-Aldrich | D2650 |
| EmbryoMAX 0.1% gelatin | EMD Millipore | ES006B |
| DPBS/Modified | HyClone | SH30028.02 |
| 0.25% Trypsin-EDTA | Life Technologies | 25200-056 |
| T25 Flask | Corning | 353108 |
| T75 flask | Corning | 353135 |
| 100-mm plate | Corning | 353003 |
| 150-mm plate | Corning | 430599 |
| 96-well plate | Corning | 3585 |
| 96-well U-bottom plate | Corning | 3799 |
| 24-well plate | Corning | 3526 |
| 50-ml reservoir | Corning | 4870 |
| 15-ml tubes | VWR International, LLC | 82050-276 |
| Primary Mouse Embryonic Fibroblasts | EMD Millipore | PMEF-NL |
| DR4 Mouse Embryonic Fibroblasts | Applied StemCell | ASF-1001 |
| Mitomycin C | Fisher BioReagents | BP25312 |
| Geneticin (G418) | Life Technologies | 11811-023 |
| Doxycycline | Fisher BioReagents | BP26531 |
| Cryotubes | Thermo Scientific | 377267 |
| Centrifuge | Eppendorf | Centrifuge 5702 |
| TC10 cell counter | Bio-Rad | |
| Counting Slides (for TC10) | Bio-Rad | 1450011 |
| Electroporation | ||
| Gene Pulser Xcell | Bio-Rad | 1652611 |
| Gene Pulser Cuvettes (4 mm gap) | Bio-Rad | 1652088 |
| Molecular Biology | ||
| Thermal Cycler | Eppendorf | Mastercylcer ep Gradient S |
| Puregene Core kit B | Qiagen | 158745 |
| Topo-TA Cloning kit | Life Technologies | 450030 |
| High Capacity cDNA synthesis kit | Applied Biosystems | 4368814 |
| NaCl | Fisher BioReagents | BP358-212 |
| 100% ethanol | Decon Laboratories, Inc. | 2716 |
| Double Processed Tissue Culture Water | Sigma-Aldrich | W3500 |
| Sau3A1 | New England BioLabs | R0169L |
| T4 DNA Ligase | New England BioLabs | M0202T |
| EcoRV | New England BioLabs | R3195S |
| 96-well Lysis Buffer (Ramires-Solis et al. 1992) | ||
| Trizma Base | Sigma-Aldrich | T1503 |
| Hydrochloric Acid | Fisher BioReagents | A144-212 |
| EDTA | Sigma-Aldrich | E5134 |
| N-Lauroylsarcosine sodium salt | Sigma-Aldrich | L5777 |
| Proteinase-K | Fisher BioReagents | BP1700 |
| Electrophoresis | ||
| Mini-Sub Cell GT | Bio-Rad | 170-4469EDU |
| LE Agarose | GeneMate | E3120500 |
| Ethidium Bromide | Fisher BioReagents | BP1302 |
| 100 BP DNA Ladder | New England BioLabs | N3231S |
| 1Kb DNA Ladder | New England BioLabs | N3232S |
| 2-log DNA Ladder | New England BioLabs | N3200L |
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