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Abstract
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Protocol
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Genetics

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Published: November 20, 2018
doi:
10.3791/58467
, , , , , , , , ,
1Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine,Hunan Agricultural University (HUNAU), 2Department of Pathology,Georgetown University Medical School, 3College of Food Science and Technology,Hunan Agricultural University (HUNAU), 4Lab of Molecular Cardiology (LMC),National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH), 5Transgenic Core,National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH)

Summary

在这里, 我们提出了一个详细的协议, 以确定同源重组事件发生在小鼠胚胎干细胞使用南方印迹和/或 pcr。利用传统的胚胎干细胞同源重组介导靶向技术生成的非肌肉肌球蛋白 ii 基因替代小鼠模型就是例证。

Abstract

相对于非目标效应和在基因组编辑的设计师核酸酶应用中插入长 dna 片段的困难问题, 胚胎茎 (es) 细胞靶向技术并没有这些缺点, 而且广泛存在用于修改动物老鼠基因组, 从大的缺失插入到单核苷酸取代。值得注意的是, 确定获得所需的 es 克隆所需的同源重组 (hr) 事件相对较少, 是一个关键步骤, 需要准确和可靠的方法。南方印迹和/或常规 pcr 经常用于这一目的。在这里, 我们描述了使用这两种方法来识别发生在小鼠 es 细胞中的 hr 事件的详细过程, 在这些事件中, 内源性 myosin 基因的目的是被编码其他非肌肉肌球蛋白重链 i (nmhc iis) 的 cDNAs 所破坏和取代。南方印迹的整个过程包括靶向载体的构建、电穿孔、药物选择、es 细胞/克隆体的扩张和储存、基因组 dna (gdna) 的制备、消化和印迹、杂交和探头的清洗, 以及 x 射线胶片上自动成像的最后一步。pcr 可直接与制备的和稀释的 gdna 一起进行。为了获得理想的结果, 应仔细规划用于南方印迹的探针和限制性酶 (re) 切割位点和 pcr 引物。虽然执行南方印迹是耗时和劳动密集型的, pcr 结果有假阳性, 但南方印迹的正确识别和 pcr 的快速筛选允许这些方法的唯一或组合应用所描述的本文在 es 细胞和转基因动物基因型的鉴定中得到了大多数实验室的广泛应用和参考。

Introduction

hr 在小鼠 es 细胞中的基因靶向技术为剖析特定基因突变 1,2的细胞后果提供了有力的工具。2007年诺贝尔生理学或医学奖 3、4 对这一技术的重要性和意义反映在它的认可上;同时, 它也代表了现代基因工程时代的到来.通过 hr 进行基因靶向的基因可以用来设计小鼠 es 细胞 6,7基因组中几乎任何改变, 从点突变到大染色体重排。众所周知, 在所谓的基因组编辑工具出现之前, 基因敲除小鼠的产生需要在 es细胞8、9、10中应用基因靶向技术。在过去20年中, 这种方法产生了 5, 000多头基因靶向小鼠, 用于人类疾病建模或研究基因功能 11。建立了全基因组淘汰赛的工作, 将基因靶向载体、针对 es 细胞克隆和活鼠分配给科学界2、12、13、14,15. 毫无疑问, 基于 es 细胞的 hrc 介导的基因靶向技术极大地促进了我们对在生理或病理背景下发挥的基因功能的了解。

由于 hr 在哺乳动物细胞16,17中是一个相对较少的事件, 重要的和下一步的基因靶向在小鼠 es 细胞是分析许多 es 菌落, 以确定几个克隆与突变产生的hr 与目标向量 18。识别人力资源事件的黄金方法包括南方印迹和 pcr19,20。这些方法的优点包括南方印迹可以正确识别有针对性的 es 克隆, 并允许研究人员分析基因目标事件的结构, 例如验证构造的单个副本插入, 而基于 pcr 的战略允许更迅速地筛选21世纪、22日的人力资源活动。尽管这些方法有缺点, 例如它们很耗时, 可能存在误报, 但它们的组合用法在识别 hr 事件 (特别是 es 细胞) 以生成转基因产品时被广泛接受和应用动物。

哺乳动物中的非肌肉肌球蛋白 ii (nm ii) 有三种异型, 每种都由两个相同的 nmhc i i 由三个不同的基因 (名为 myosin、myosin 和 myosin) 和两对光链编码, 称为 nm-ii-a、ii-b 和 ii-c23。先前的研究表明, 至少 nm ii-a 和 ii-b 的异形对小鼠的发育至关重要, 因为这些同态的体内消融会导致胚胎性 24,25, 26。为了规避这一问题, 并在小鼠发育的后期阶段对 nm ii-a 和 ii-b 的特定于于等结构的功能有新的见解, 采用了基于 es 细胞的 hr 介导基因靶向技术的基因替代策略。生成一系列鼠标模型27。在确定所需 es 克隆体的过程中, 采用了南方印迹和 pcr 方法, 证明了这些方法的有效性和可靠性。

本文旨在提供南方印迹和 pcr 的详细描述, 包括靶向载体、探针和引物的设计, 实验的执行, 以及通过识别 hr 事件对结果进行分析。发生在 es 细胞中, 用于创建基因替代 nm ii 小鼠模型和代表性数据。这里介绍的这两种方法的方案也可以用来识别转基因细胞或动物的基因型。

Protocol

1. pcr 的目标结构、南球探针和引物的设计 选择 myh9 基因的第一个编码外显子 (exon 2), 用于在此处报告的敲击/敲入应用中中断或插入。 从genome.ucsc.edu网站上检索 myh9 外显子2周围的5-kb 上游和5-kb 下游 dna 序列。 分析在这个10-kb 区域减少1-2 的酶 (res) 的限制性消化模式, 使用 pdraw 软件来确定合适的 re (s) 来消化用于南方印迹的基因组 dna。注: dra i 满足此要求, 并?…

Representative Results

本文介绍了一种详细的南方印迹和 pcr 协议, 该协议利用基于 es 细胞的 hr 介导靶向技术, 用于识别小鼠 es 细胞中发生的 hr 事件, 用于生成 nm ii 基因替代小鼠模型。尽管南方印迹和 pcr 以及传统的基因靶向技术已经广泛使用了几十年, 但它们的成功应用需要仔细规划。至少需要考虑这些方面: 长臂和短臂的长度、探针的位置和长度、切割基因组 dna 的合适 res 以及 pcr 的引物, ?…

Discussion

目前, 用于基因组编辑的设计核酸酶由于其目标外效应和插入 dna 片段30、31的困难问题, 仍不能取代基于 es 细胞的基因靶向技术。作为识别发生在小鼠 es 细胞中的 hr 事件的黄金方法, 本报告为该领域提供了一个详细的南方印迹和 pcr 协议。我们通过分析一系列构造所针对的鼠标 es 细胞中的单个克隆来验证这些方法的可靠性。这些方法确定的所需 es 克隆?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家自然科学基金总计划 (31571432)、中国省人类自然科学基金会 (赠款号 2015jc3097) 和湖南省教育局研究基金会的支持。中国省 (批准号: 15k054)。

Materials

BAC CLONE BACPAC Resources Center (BPRC) bMQ-330E21
QIAGEN Large-Construct Kit QIAGEN 12462
QIAquick Gel Extraction Kit QIAGEN 28704
QIAquick PCR Purification Kit QIAGEN 28104
QIAprep Spin Miniprep Kit QIAGEN 27104
QIAGEN Plasmid Plus Maxi Kit QIAGEN 12963
PfuUltra High-Fidelity DNA Polymerase Agilent 600382
T-easy vector Promega A1360
Nuclei Lysis Solution Promega A7941
Protein Precipitation Solution Promega A7951
DNA Denaturing Solution VWR 351-013-131
DNA Neutralizing Solution VWR 351-014-131
Ready-To-Go DNA Labeling Beads (-dCTP) VWR 27-9240-01
UltraPure SSC, 20X Thermo Fisher 15557036
UltraPur Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) Thermo Fisher 15593031
G418 Thermo Fisher 10131035
Salmon Sperm DNA Solution Thermo Fisher 15632011
Platinu Taq DNA Polymerase High Fidelity Thermo Fisher 11304029
Not I Thermo Scientific ER0592
Dra I Thermo Scientific ER0221
EcoR I Thermo Scientific ER0271
Ganciclovir Sigma G2536
Whatman TurboBlotter Transfer System, Large Kits Fisher Scientific 09-301-188
[α32P]dCTP PerkinElmer NEG013H100UC
ProbeQuan G-50 Micro Columns GE Healthcare 28-9034-08
Hybrisol I Hybridization Solution Millipore S4040
Kodak X-Ray Film Z&Z Medical 844 5702
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Tags

Homologous RecombinationMouse Embryonic Stem CellsSouthern BlottingPolymerase Chain ReactionMolecular BiologyCellular BiologyGenetically Modified CellsGDNA DigestionAgarose Gel ElectrophoresisDNA TransferDNA LabelingDNA Hybridization
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Zhou, D., Tan, L., Li, J., Liu, T., Hu, Y., Li, Y., Kawamoto, S., Liu, C., Guo, S., Wang, A. Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction. J. Vis. Exp. (141), e58467, doi:10.3791/58467 (2018).
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