使用冷冻胚胎进行转基因小鼠高效生产
1Max Planck Florida Institute for Neuroscience, 2Life Science Research Center,University of Toyama, 3Department of Biochemical Engineering, Graduate School of Science and Engineering,Yamagata University, 4Graduate School of Innovative Life Science,University of Toyama, 5Department of Biochemistry, Faculty of Pharmacy,Cairo University, 6Division of Regenerative Medicine, Center for Molecular Medicine,Jichi Medical University, 7Division of Stem Cell Research and Drug Development, Center for Development of Advanced Medical Technology,Jichi Medical University, 8Synthetic Biology Division, Research Center for Advanced Science and Technology,University of Tokyo, 9Institute for Advanced Biosciences,Keio University, 10Graduate School of Media and Governance,Keio University, 11Department of Molecular Oncology, Graduate School of Medicine,Chiba University, 12Department of Biological Sciences, School of Science,University of Tokyo, 13College of Arts and Sciences,University of Tokyo
Summary
在这里，我们提出了一种用于冷冻保存单细胞胚胎的改良方法，以及一种用于使用冷冻解冻胚胎和电穿孔以有效生成转基因小鼠的协议。
Abstract
转基因小鼠的使用已成为理解基因功能和破译人类疾病的基本机制的关键。CRISPR/Cas9 系统允许研究人员以前所未有的效率、保真度和简单性修改基因组。利用这项技术，研究人员正在寻求一种快速、高效和简单的生成转基因小鼠的协议。在这里，我们介绍了一种改进的单细胞胚胎冷冻保存方法，该方法导致冷冻解冻胚胎的发育速度更高。通过将其与优化的电镀条件相结合，该协议允许在短时间内生成高效率和低镶嵌率的挖空和敲敲小鼠。此外，我们逐步解释我们优化的协议，包括CRISPR试剂制备、体外受精、一细胞胚胎的冷冻保存和解冻、CRISPR试剂的电穿孔、小鼠的生成和创始人的基因分型。使用这种协议，研究人员应该能够以无与伦比的轻松、速度和效率制备转基因小鼠。
Introduction
集群定期间隔短蛋白重复（CRISPR）/CRISPR相关蛋白9（Cas9）系统是一个科学突破，提供了前所未有的有针对性的修改在基因组1。CRISPR/Cas9系统由Cas9蛋白和引导RNA（gRNA）组成，含有两个分子成分：一个目标特异性CRISPRRNA（crRNA）和一个转激活的CRISPRRNA（气管RNA）2。gRNA将Cas9蛋白定向到基因组中的特定位点，20个核苷酸与crRNA互补，并靠近原间隔体相邻的图案（PAM）。Cas9蛋白与目标序列结合，并诱导双绞线断裂（DSBs），由容易出错的非同源端连接（NHEJ）或高保真同源定向修复（HDR）3，4，5修复。3,4,5NHEJ 导致插入或/和删除（indels），因此在编码序列被靶向时导致功能基因丧失。HDR导致在包含同源序列33，4，54,5的修复模板的情况下进行精确的基因组编辑。NHEJ 和 HDR 已分别用于生成敲敲小鼠和敲敲小鼠。
虽然CRISPR/Cas9系统显著加速了具有卓越功效和保真度的转基因小鼠的生成，但应用这些方法的科学家经常遇到技术挑战。首先，常规协议需要微注射，将CRISPR编辑工具引入受精卵66、77的排卵核。这种技术非常耗时，通常需要广泛的培训。因此，几个组取代显微注射与电穿孔898，9，10，11，12，13。,10,11,12,13,然而，在早期的电穿孔协议中，新鲜胚胎被用于电镀。这就引起了另一个问题，因为每次实验前准备新鲜胚胎是很困难的。
最近我们和其他人将冷冻解冻胚胎和电穿孔结合起来进行基因组编辑，这为转基因小鼠15、16,16的生成提供了便利。该协议使没有高级胚胎操作技能的研究人员能够高效快速生成人类疾病的动物模型。该协议还大大减少了在生成转基因小鼠方面的实际挑战，如创始人16中的遗传异质性。为了克服马赛克，我们在胚胎解冻后1小时内对CRISPR试剂进行电镀，以确保在基因组首次复制之前进行编辑。另一个改进包括使用Cas9蛋白代替Cas9 mRNA来减少不受欢迎的马赛克17。此外，我们开发了一种单细胞胚胎冷冻保存的最佳方法，将发育速率提高到双细胞第16阶段：使用胎儿牛血清（FBS）可以显著提高受精后冷冻解冻卵母细胞的存活率，或许通过使冷冻解冻未受精的卵母细胞更具弹性的18。
在这里，我们提出了一个全面的协议，为使用冷冻解冻胚胎的转基因小鼠的生成，包括单细胞C57BL/6J胚胎冷冻保存的改良方法。它包括1gRNA设计、CRISPR试剂制备和组装;2） 试管婴儿、冷冻保存和单细胞胚胎解冻;3） 将CRISPR试剂电化成冷冻解冻的胚胎;4） 胚胎移植成伪怀孕雌性小鼠的卵子;和5） F0创始人动物的基因分型和序列分析。
Protocol
本研究中执行的所有动物护理和程序都是根据《实验室动物护理和使用指南》的规则和条例进行的。实验方案经富山大学动物动物委员会、东京大学、吉池大学和马克斯·普朗克佛罗里达神经科学研究所批准。有关所有试剂的信息都显示在材料表中。 1. CRISPR 试剂设计 crRNA 设计 访问 CRISPR 直接19 （https://crispr.dbcls.jp/），以设计具有?…
Representative Results
我们改进的单细胞胚胎冷冻保存方法，包括在含有20S的HTF中孵育10分钟，然后采用1 M DMSO和DAP213溶液进行冷冻保存，提高了冷冻解冻胚胎进入双细胞阶段的发育速率（图1，p= 0.009，学生的T-test）。冷冻解冻胚胎用于生产转基因小鼠，电穿孔条件得到优化：使用《议定书》部分所述的电穿孔器，使用25 V和3 ms脉冲和97 ms间隔的5次重复。该协议的适用性通过白化剂酪氨酸酶基因?…
Discussion
所述协议允许生成高效和低马赛克率的转基因小鼠（表1）。它使没有高级胚胎操作技能的研究人员能够轻松培育出突变小鼠，因为它利用了生殖工程和基因组编辑技术的最新和最有用的进步：CRISPR/Cas9核苷酸蛋白（RNP）和电穿孔进入冷冻解冻的胚胎。这些进步促进了转基因小鼠的生成，并加快了生产速度。如图3所示，生成转基因小鼠需要+4周。与其他采用类似?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢希托米·萨瓦达和伊丽莎白·加西亚的动物护理。这项工作得到了KAKENHI（15K20134，17K11222，16H06276和16K06276和16K01946）和厚金研究补助金（至H.N.）和吉奇医科大学青年调查员奖（至H.U.）的支持。大须贺托希米奖学金基金会支持M.D.
Materials
| 0.25 M Sucrose | ARK Resource Co., Ltd. (Kumamoto, Japan) | SUCROSE | |
| 1 M DMSO | ARK Resource Co., Ltd. (Kumamoto, Japan) | 1M DMSO | |
| Butorphanol | Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) | Vetorphale 5mg | |
| Cas9 protein: Alt-R® S.p. HiFi Cas9 Nuclease 3NLS | Integrated DNA Technologies, Inc. (Coralville, IA) | 1081060 | |
| C57BL/6J mice | Japan SLC (Hamamatsu, Japan) | N/A | |
| DAP213 | ARK Resource Co., Ltd. (Kumamoto, Japan) | DAP213 | |
| FBS | Sigma-Aldrich, Inc. (St. Louis, MO) | ES-009-C | |
| hCG | MOCHIDA PHARMACEUTICAL CO., LTD (Tokyo, Japan) | HCG Mochida 3000 | |
| HTF | ARK Resource Co., Ltd. (Kumamoto, Japan) | HTF | |
| ICR mice | Japan SLC (Hamamatsu, Japan) | N/A | |
| Isoflurane | Petterson Vet Supply, Inc. (Greeley, CO) | 07-893-1389 | |
| KSOM | ARK Resource Co., Ltd. (Kumamoto, Japan) | KSOM | |
| LN2 Tank | Chart Industries (Ball Ground, GA) | XC 34/18 | |
| M2 | ARK Resource Co., Ltd. (Kumamoto, Japan) | M2 | |
| Medetomidine | Nippon Zenyaku Kogyo Co.,Ltd. (Koriyama, Japan) | 1124401A1060 | |
| Microscope | Nikon Co. (Tokyo, Japan) | SMZ745T | |
| Midazolam | Sandoz K.K. (Tokyo, Japan) | 1124401A1060 | |
| Nuclease free buffer | Integrated DNA Technologies, Inc. (Coralville, IA) | 1072570 | |
| Nucleospin DNA extraction kit | Takara Bio Inc (Kusatsu, Japan) | 740952 .5 | |
| One-hole slide glass | Matsunami Glass Ind., Ltd. (Kishiwada, Japan) | S339929 | |
| One-step type Electroporator | BEX Co., Ltd. (Tokyo, Japan) | CUY21EDIT II | |
| Paraffin Liquid | NACALAI TESQUE Inc. (Kyoto, Japan) | SP 26137-85 | |
| Platinum plate electrode | BEX Co., Ltd. (Tokyo, Japan) | LF501PT1-10, GE-101 | |
| PMSG | ASKA Animal Health Co., Ltd (Tokyo, Japan) | SEROTROPIN 1000 | |
| Povidone iodide | Professional Disposables International, Inc. (Orangeburg, NY) | C12400 | |
| Reduced-Serum Minimal Essential Medium: OptiMEM I | Sigma-Aldrich, Inc. (St. Louis, MO) | 22600134 | |
| Two-step type Electroporator | Nepa Gene Co., Ltd. (Ichikawa, Japan) | NEPA21 |
References
- Wang, H., et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering. Cell. 153 (4), 910-918 (2013).
- Jinek, M., et al. A programmable dual-RNA – guided DNA endonuclease in adaptive bacterial immunity. Science. 337 (6096), 816-822 (2012).
- Mali, P., et al. RNA-guided human genome engineering via Cas9. Science. 339 (6121), 823-826 (2013).
- Cong, L., et al. Multiplex genome engineering using CRISPR/VCas systems. Science. 339 (6121), 819-823 (2013).
- Doudna, J. A., Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science. 346 (6213), 1258096 (2014).
- Mashiko, D., et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Scientific Reports. 3, 3355 (2013).
- Yen, S. T., et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Biologie du développement. 393 (1), 3-9 (2014).
- Kaneko, T., Mashimo, T. Simple genome editing of rodent intact embryos by electroporation. PLoS ONE. 10 (11), 1-7 (2015).
- Qin, W., et al. Efficient CRISPR/cas9-mediated genome editing in mice by zygote electroporation of nuclease. Génétique. 200 (2), 423-430 (2015).
- Kaneko, T., Sakuma, T., Yamamoto, T., Mashimo, T. Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Scientific Reports. 4, 6382 (2014).
- Hashimoto, M., Takemoto, T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Scientific Reports. 5, 11315 (2015).
- Chen, S., Lee, B., Lee, A. Y. F., Modzelewski, A. J., He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. Journal of Biological Chemistry. 291 (28), 14457-14467 (2016).
- Modzelewski, A. J., et al. Efficient mouse genome engineering by CRISPR-EZ technology. Nature Protocols. 13 (6), 1253-1274 (2018).
- Teixeira, M., et al. Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing. Scientific Reports. 8, 474 (2018).
- Nakagawa, Y., et al. Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnology. 15 (1), 1-10 (2015).
- Darwish, M., et al. Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain. Journal of Neuroscience Methods. 317, 149-156 (2019).
- Hashimoto, M., Yamashita, Y., Takemoto, T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Biologie du développement. 418 (1), 1-9 (2016).
- Sakamoto, W., Kaneko, T., Nakagata, N. Use of frozen-thawed oocytes for efficient production of normal offspring from cryopreserved mouse spermatozoa showing low fertility. Comparative Medicine. 55 (2), 136-139 (2005).
- Naito, Y., Hino, K., Bono, H., Ui-Tei, K. CRISPRdirect: Software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics. 31 (7), 1120-1123 (2015).
- Torres-Perez, R., Garcia-Martin, J. A., Montoliu, L., Oliveros, J. C., Pazos, F. WeReview: CRISPR Tools-Live Repository of Computational Tools for Assisting CRISPR/Cas Experiments. Bio-ingénierie. 6 (3), 63 (2019).
- Okamoto, S., Amaishi, Y., Maki, I., Enoki, T., Mineno, J. Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Scientific Reports. 9, 4811 (2019).
- Takeo, T., Nakagata, N. Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PLoS ONE. 10 (5), 1-11 (2015).
- Wuri, L., Agca, C., Agca, Y. Euthanasia via CO2 inhalation causes premature cortical granule exocytosis in mouse oocytes and influences in vitro fertilization and embryo development. Molecular Reproduction and Development. 86 (7), 825-834 (2019).
- Nakagata, N. High survival rate of unfertilized mouse oocytes after vitrification. Journal of Reproduction and Fertility. 87 (2), 479-483 (1989).
- Dehairs, J., Talebi, A., Cherifi, Y., Swinnen, J. V. CRISP-ID: Decoding CRISPR mediated indels by Sanger sequencing. Scientific Reports. 6, 28973 (2016).
- Nakagawa, Y., Sakuma, T., Takeo, T., Nakagata, N., Yamamoto, T. Electroporation-mediated genome editing in vitrified/warmed mouse zygotes created by ivf via ultra-superovulation. Experimental Animals. 67 (4), 535-543 (2018).
- Nakajima, K., Nakajima, T., Takase, M., Yaoita, Y. Generation of albino Xenopus tropicalis using zinc-finger nucleases. Development Growth and Differentiation. 54 (9), 777-784 (2012).
- Hur, J. K., et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nature Biotechnology. 34, 807-808 (2016).
- Dumeau, C. E., et al. Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1. Transgenic Research. 28 (5-6), 525-535 (2019).
- Chen, S., et al. CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection. Cell Reports. 27 (13), 3780-3789 (2019).
- Mizuno, N., et al. Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector. iScience. 9, 286-297 (2018).
- Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. Highly Efficient RNA-guide genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research. 24 (6), 1012-1019 (2014).
- Vakulskas, C. A., et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nature Medicine. 24 (8), 1216-1224 (2018).
- Rodriguez-Rodriguez, J. A., et al. Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion. Current Biology. 28 (21), 3422-3429 (2018).
- Smits, A. H., et al. Biological Plasticity Rescues Target Activity in CRISPR Knockouts. Nature Methods. 16, 1087-1093 (2019).
Citer Cet Article
Nishizono, H., Darwish, M., Uosaki, H., Masuyama, N., Seki, M., Abe, H., Yachie, N., Yasuda, R. Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice. J. Vis. Exp. (158), e60808, doi:10.3791/60808 (2020).