Summary

通过显微注射卵母细胞生成转基因小鼠

Published: June 15, 2017
doi:

Summary

小鼠卵母细胞的显微注射通常用于经典转基因( 转基因的随机整合)和CRISPR介导的基因靶向。该协议审查显微注射的最新发展,特别强调质量控制和基因分型策略。

Abstract

使用转基因小鼠对生理和病理体内过程的研究有显着的贡献。将DNA表达构建体的原核注射到受精卵中仍然是产生用于过表达的转基因小鼠的最常用技术。随着用于基因靶向的CRISPR技术的引入,原核注入受精卵母细胞已经扩展到敲除和敲入小鼠的产生。这项工作描述了用于注射的DNA的制备和用于基因靶向的CRISPR指南的产生,特别强调质量控制。识别潜在创始人所需的基因分型程序至关重要。本文介绍了利用CRISPR“复用”能力的创新型基因分型策略。还概述了外科手术。一起,协议的步骤将允许生成gen以及随后建立的大量研究领域的小鼠集落,包括免疫学,神经科学,癌症,生理学,发育等。

Introduction

在脊椎动物和无脊椎动物中的动物模型已经有助于检查人类病情如阿尔茨海默病1,2的病理生理学。他们也是寻找疾病修饰剂的最宝贵的工具,并最终开发出新的治疗策略,希望得到治愈。虽然每个模型都有固有的局限性,但是使用动物作为整个系统模型对于生物医学研究至关重要。这是因为组织培养不能完全模拟代谢和复杂的生理环境。

迄今为止,鼠标仍然是用于遗传操作的最常见的哺乳动物物种,因为它具有几个优点。与疾病相关的生理过程和基因在小鼠和人之间是高度保守的。小鼠是第一个拥有全基因组测序的哺乳动物(2002年),人类基因组前一年我(2003)。除了丰富的遗传信息,小鼠具有良好的育种能力,快速的开发周期(从受精到断奶6周)和合理的大小。所有这些优点,加上生理指标,如独特的外套颜色(交叉策略所需),使得鼠标成为遗传操作的有吸引力的模型。值得注意的是,在现代遗传学的很早的时代,格雷戈尔·门德尔(Gregor Mendel)开始对老鼠进行植物移植3

基因转移技术导致在三十年前第一代转基因小鼠的产生4 ,最初使用病毒递送。然而,研究人员很快意识到,小鼠转基因的主要挑战之一是无法控制外源DNA的命运。因为转基因向小鼠卵母细胞的病毒递送导致随机地整合到基因组中的多个拷贝,这是可能的建立后续转基因株系的限制。

当Gordon 等人通过显微注射产生第一个转基因小鼠系列5,6 。这开始了重组DNA技术的时代,影响显微注射结局的参数已被广泛研究7 。虽然显微注射不允许控制转基因的整合位点(最终导致每个创始人小鼠的特异性表达水平),但是原核显微注射的主要优点仍然是形成连续体( 即,转基因的多个拷贝的阵列,在基因组整合前5) 。多年来已经使用这种特征来建立数千个过表达感兴趣的基因的转基因小鼠品系。从那时起,转基因,a生物体基因组的人工修饰已广泛用于鉴定单一基因在疾病发生中的作用。

当马里奥·卡佩奇奇成功地破坏了小鼠中的单个基因时,达到了操纵小鼠基因组的另一个关键成果,打开了基因靶向时代8 。然而,基于ES细胞的基因靶向快速出现主要缺点,包括培养ES细胞的挑战,嵌合体的程度有所不同,以及进程的长度( 12-18个月,最小化获得小鼠) 。

最近,已经出现了诸如工程内切核酸酶( 例如锌指核酸酶(ZFN),转录激活物样效应核酸酶(TALEN))和聚集的定期交织的短回文重复序列(CRISPR / Cas9)的新技术的进展,作为替代方法加速麦克风中基因靶向过程e 9,10 。这些核酸内切酶可以通过显微注射容易地注入小鼠卵母细胞,从而在短短6周内产生基因靶向的小鼠。

自从关于使用CRISPR进行基因组编辑的第一份报告11以来,这种细菌适应性免疫系统由于具有许多优点而取代了ZFN和TALEN,包括易于合成和一次靶向多个基因座的能力(简称“复用” “)。 CRISPR首先用于小鼠12中的基因靶向,并已被应用于从植物到人类的无数种类13,14 。迄今为止,还没有关于抗CRISPR基因组编辑的单一物种的报道。

产生转基因小鼠的两个主要限制步骤是注射卵母细胞和再植入的这些卵母细胞变成假怀孕的雌性。尽管我们已经描述了这种技术15和其他16 ,但是近来在小鼠胚胎学和基因转移技术方面的技术改进已经改变了生产转基因小鼠的过程。这里将描述这些改进。

Protocol

所有程序已获得新南威尔士大学动物护理与伦理委员会的批准。 1.准备转基因(随机整合) 分析琼脂糖凝胶电泳。 根据制造商的建议,使用合适的酶(1小时孵育)或快速消化酶(15至30分钟孵育)在热循环仪中消化质粒以切割转基因(参见图2A及其图例)。 加入1%三醋酸乙二胺四乙酸(EDTA)(TEA)琼脂糖凝胶,用0.5-1…

Representative Results

下面描述了在随机整合和CRISPR介导的基因靶向的情况下显微注射的工作流程( 图1 )。 图1:产生转基因小鼠的典型工作流程。对于随机整合,将纯化的转基因注射入受精卵母细胞的原核,然后输卵管转移到插入的寄养雌性中?…

Discussion

协议中的关键步骤

已知基因修饰的小鼠的产生在技术上是具有挑战性的。然而,这里提出的协议是一种优化和简化的方法,可以在记录时间内掌握和排除故障。成功完成技术需要两个步骤。首先,不用氯化镁(MgCl 2 )可以实现线性DNA模板的合成(用于合成sgRNA)。然而,强烈建议系统地将MgCl 2加入到主混合物中,因为在没有MgCl 2的情况下经常防止正向引…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢动物设施(BRC)的工作人员不断的支持。这项工作由国家卫生和医学研究理事会和澳大利亚研究理事会资助。

Materials

Micropipette 0.1-2.5 ul Eppendorf 4920000016
Micropipette 2-20 ul Eppendorf 4920000040
Micropipette 20-200 ul Eppendorf 4920000067
Micropipette 100-1000 ul Eppendorf 4920000083
Molecular weight marker  Bioline BIO-33025 HyperLadder 1kb
Molecular weight marker  Bioline BIO-33056 HyperLadder 100 bp
Agarose Bioline BIO-41025
EDTA buffer Sigma-Aldrich 93296 10x – Dilute to 1x
Ethidium bromide Thermo Fisher Scientific 15585011
SYBR Safe gel stain Invitrogen S33102
Gel extraction kit Qiagen 28706
PCR purification kit (Qiaquick) Qiagen 28106
Vacuum system (Manifold) Promega A7231
Nuclease-free microinjection buffer  Millipore MR-095-10F
Ultrafree-MC microcentrifuge filter  Millipore UFC30GV00
Cas9 mRNA Sigma-Aldrich CAS9MRNA
CRISPR expressing plasmid (px330) Addgene 42230
Nuclease free water Sigma-Aldrich W4502
Phusion polymerase New England Biolabs M0530L
T7 Quick High Yield RNA kit New England Biolabs E2050S
RNA purification spin columns (NucAway) Thermo Fisher Scientific AM10070
ssOligos Sigma-Aldrich OLIGO STANDARD
Donor plasmid Thermo Fisher Scientific GeneArt
Hyaluronidase Sigma-Aldrich H3884
KSOMaa embryo culture medium Zenith Biotech  ZEKS-100
Mineral oil Zenith Biotech  ZSCO-100
M2 Medium Sigma-Aldrich M7167
Cytochalasin B Sigma-Aldrich C6762
Mouthpiece Sigma-Aldrich A5177
Glass microcapillaries Sutter Instrument BF100-78-10
Proteinase K Applichem A3830.0100
Dumont #5 forceps Fine Science Tools  91150-20
Iris scissors Fine Science Tools  91460-11
Vessel clamp Fine Science Tools  18374-43
Wound clips  Fine Science Tools  12040-01
Clips applier  Fine Science Tools  12018-12
Micro-scissors  Fine Science Tools  15000-03
Cauterizer Fine Science Tools  18000-00
Non-absorbable surgical sutures (Ethilon 3-0) Ethicon 1691H
5% CO2 incubator MG Scientific Galaxy 14S
Spectrophotometer Thermo Fisher Scientific Nanodrop 2000c
Thermocycler Eppendorf 6321 000.515
Electrophoresis set up BioRad 1640300
UV Transilluminator BioRad 1708110EDU
Thermocycler Eppendorf 6334000069
Stereoscopic microscope Olympus SZX7
Inverted microscope Olympus IX71
2x Micromanipulators Eppendorf 5188000.012
Oocytes manipulator Eppendorf 5176000.025
Microinjector (Femtojet) Eppendorf 5247000.013
Mice C57BL/6J strain Australian BioResources C57BL/6JAusb 

References

  1. Ittner, L. M. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer’s disease mouse models. Cell. 142 (3), 387-397 (2010).
  2. Ittner, A. Site-specific phosphorylation of tau inhibits amyloid-beta toxicity in Alzheimer’s mice. Science. 354 (6314), 904-908 (2016).
  3. Marantz Henig, R. . The Monk in the Garden. , (2000).
  4. Jaenisch, R., Mintz, B. Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc Natl Acad Sci U S A. 71 (4), 1250-1254 (1974).
  5. Gordon, J. W., Ruddle, F. H. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science. 214 (4526), 1244-1246 (1981).
  6. Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., Ruddle, F. H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 77 (12), 7380-7384 (1980).
  7. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A. 82 (13), 4438-4442 (1985).
  8. Capecchi, M. R. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet. 6 (6), 507-512 (2005).
  9. Carbery, I. D. Targeted genome modification in mice using zinc-finger nucleases. 유전학. 186 (2), 451-459 (2010).
  10. Sung, Y. H. Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol. 31 (1), 23-24 (2013).
  11. Jinek, M. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337 (6096), 816-821 (2012).
  12. Wang, H. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 153 (4), 910-918 (2013).
  13. Kang, X. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J Assist Reprod Genet. 33 (5), 581-588 (2016).
  14. Liang, P. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 6 (5), 363-372 (2015).
  15. Ittner, L. M., Götz, J. Pronuclear injection for the production of transgenic mice. Nat Protoc. 2 (5), 1206-1215 (2007).
  16. Yang, H., Wang, H., Jaenisch, R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat Protoc. 9 (8), 1956-1968 (2014).
  17. . Resuspension Calculator Available from: https://sg.idtdna.com/calc/resuspension/ (2017)
  18. Auerbach, A. B. Strain-dependent differences in the efficiency of transgenic mouse production. Transgenic Res. 12 (1), 59-69 (2003).
  19. Merriman, J. A., Jennings, P. C., McLaughlin, E. A., Jones, K. T. Effect of aging on superovulation efficiency, aneuploidy rates, and sister chromatid cohesion in mice aged up to 15 months. Biol Reprod. 86 (2), 49 (2012).
  20. Nakagawa, Y., et al. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice. Biol Open. 5 (8), 1142-1148 (2016).
  21. Byers, S. L., Wiles, M. V., Dunn, S. L., Taft, R. A. Mouse estrous cycle identification tool and images. PLoS One. 7 (4), e35538 (2012).
  22. Whitten, W. K. Modification of the oestrous cycle of the mouse by external stimuli associated with the male. J Endocrinol. 13 (4), 399-404 (1956).
  23. Ye, S., Dhillon, S., Ke, X., Collins, A. R., Day, I. N. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res. 29 (17), E88 (2001).
  24. Yoshimi, K., et al. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun. 7, 10431 (2016).
  25. Suzuki, K., et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. , (2016).
  26. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 21, 70-71 (1999).
  27. Liu, X., et al. A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling. J Cell Biol. 130 (1), 227-237 (1995).
  28. Ke, Y. D. Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS. Acta Neuropathol. 130 (5), 661-678 (2015).
  29. Auerbach, A. B. Production of functional transgenic mice by DNA pronuclear microinjection. Acta Biochim Pol. 51 (1), 9-31 (2004).
  30. Iyer, V., et al. Off-target mutations are rare in Cas9-modified mice. Nat Methods. 12 (6), 479 (2015).
  31. Zhou, Y. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature. 509 (7501), 487-491 (2014).
  32. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Avarbock, M. R. Translation of globin messenger RNA by the mouse ovum. Nature. 283 (5746), 499-501 (1980).
  33. Pinkert, C. A., Irwin, M. H., Johnson, L. W., Moffatt, R. J. Mitochondria transfer into mouse ova by microinjection. Transgenic Res. 6 (6), 379-383 (1997).
  34. Biggers, J. D., Summers, M. C. Choosing a culture medium: making informed choices. Fertil Steril. 90 (3), 473-483 (2008).
  35. Nakagata, N. Embryo transfer through the wall of the fallopian tube in mice. Jikken Dobutsu. 41 (3), 387-388 (1992).
  36. Truett, G. E. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques. 29 (1), 52-54 (2000).
  37. Richardson, C. D., Ray, G., DeWitt, M. A., Curie, G. L., Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 34 (3), 339-344 (2016).
  38. Delerue, F., Ittner, L. M. Genome Editing in Mice Using CRISPR/Cas9: Achievements and Prospects. Clon. Transgen. 4, (2015).

Play Video

Cite This Article
Delerue, F., Ittner, L. M. Generation of Genetically Modified Mice through the Microinjection of Oocytes. J. Vis. Exp. (124), e55765, doi:10.3791/55765 (2017).

View Video