Summary

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

Published: November 11, 2015
doi:

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)。 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

1.基因被困胚胎干细胞库建设使用piggyBac转座子准备原代小鼠胚胎成纤维细胞(PMEF)作为馈线ES细胞培养在37℃水浴中解冻丝裂霉素C灭活PMEF(5.0×10 6)中的一个小瓶内。转移细胞到5ml ES细胞培养基(含有15%FBS的DMEM,1000单位/ ml白血病抑制因子,为100μM非必需氨基酸,2mM谷氨酰胺,55μM2-巯基乙醇,和25单位/ ml青霉素/链霉素),和离心机在100×g离心5分钟。 吸出?…

Representative Results

在典型的诱变实验中,转染由共3×10 7个ES细胞用PB-UPA和MPB转座的。在两个独立的实验中产生的基因捕获的ES细胞的数目总结在表1中。基因包封的效率是约0.04%。合并后的基因陷阱库包含22,400独立的突变体,小鼠基因组的约一个全覆盖(23,000编码基因)。过度的覆盖可以通过反复诱变产生更多的突变体和/或大规模的进程来实现。 因为基因陷阱库包含随机?…

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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Cite This Article
Ludwig, M., Kitzenberg, D., Chick, W. S. Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells. J. Vis. Exp. (105), e53062, doi:10.3791/53062 (2015).

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