ここではユーザーの皆様に役に立ちそうな実験プロトコルが載っている文献をご紹介します。 文献には NBRP ネッタイツメガエルで開発された遺伝子破壊関係技術のものも含まれます。また、当リソースセンターが提供したリソースを用いた文献も含まれます。

NBRP ネッタイツメガエル事業により開発されたプロトコルの文献
当 NBRP リソースセンターが提供したリソースを用いた文献
上記以外のツメガエルを用いた文献



CRISPR/Cas9

(1) 基本技術

PCR を用いた sgRNA の作成、他


T. Nakayama, I. L. Blitz, M. B. Fish, A. O. Odeleye, S. Manohar, K. W. Y. Cho. And R. M. Grainger. Cas9-Based Genome Editing in Xenopus tropicalis. Methods in Enzymology 2014; 546: 355-375



(2) 効率上昇

oocyte injection & host transfer 法によりF0でキメラでない hetero KO 作成


Y. Aslan, E. Tadjuidje, A. M. Zorn and S.W. Cha. High efficiency non-mosaic CRISPR mediated knock-in and mutations in F0 Xenopus. Development 2017; 144(15): 2852-2858



(3) 組織特異(優先)的発現

生殖細胞 (Leapfrogging): ノックアウト致死遺伝子のノックアウトホモ個体を得る


I. L. Blitz, M. B. Fish and K. W. Y. Cho. Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes. Development 2016; 143: 2868-2875



腎臓


B. D. DeLay, M. E. Corkins, H. L. Hanania, M. Salanga, J. M. Deng, N. Sudou, M. Taira, M. Horb and R. K. Miller. Tissue-specific gene inactivation in Xenopus laevis: knockout of lhx1 in the kidney with CRISPR/Cas9. Genetics 2018; 208(2): 673-686



(4) ノックイン

ssODN


Y. Aslan, E. Tadjuidje, A. M. Zorn and S.W. Cha. High efficiency non-mosaic CRISPR mediated knock-in and mutations in F0 Xenopus. Development 2017; 144(15): 2852-2858



ssODN

K. Yoshimi, Y. Kunihiro, T. Kaneko, H. Nagahora, B. Voigt and T. Mashimo. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nature COMMUNICATIONS 2016; 7: 10431




homology-independent targeted integration (HITI)

K. Suzuki et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 2016 December; 540:  144-149




split GFP

D. Kamiyama, S. Sekine, B. Barsi-Rhyne, J. Hu, B. Chen, L. A. Gilbert, H. Ishikawa, M. D. Leonetti, W. F. Marshall, J. S. Weissman and B. Huang. Versatile protein tagging in cells with split fluorescent protein. Nature COMMUNICATIONS 2016; 7: 11046




CRIS-PITCh


S. Nakade, T. Tsubota, Y. Sakane, S. Kume, N. Sakamoto, M. Obara, T. Daimon, H. Sezutsu, T. Yamamoto, T.Sakuma and K. T. Suzuki. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nature COMMUNICATIONS 2014; 5: 5560




short homologous sequences (10–40 bp)

Y. Hisano, T. Sakuma, S. Nakade, R. Ohga, S. Ota, H. Okamoto, T. Yamamoto & A. Kawahara. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Scientific Reports 2015; 5: 8841



(5) その他

塩基修飾


DS. Park, M. Yoon, J. Kweon, AH. Jang, Y. Kim, and SC. Choi. Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos. Molecules and Cells 2017; 40(11): 823~827




TALEN

(1) 基本技術

TALEN構築原法

T. Cermak, E. L. Doyle, M. Christian, L. Wang, Y. Zhang, C. Schmidt, J. A. Baller, N. V. Somia, A. J. Bogdanove and D. F. Voytas. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research 2011; 39(12): e82




TALEN構築を行うための重要なポイントを紹介


K. Nakajima, Y. Nakai, M. Okada and Y. Yaoita. Targeted gene disruption in the Xenopus tropicalis genome using designed TALE nucleases. Zoological Science 2013 June; 30(6): 455-460




ネッタイツメガエルにおいて様々な TALEN を比較し、最適な TALEN を明らかに


K. Nakajima and Y. Yaoita. Comparison of TALEN scaffolds in Xnopus tropicalis. Biology Open 2013 November; 2: 1364-1370




TALENによるノックアウトのタイムコース


K. Nakajima and Y. Yaoita. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis. Biology Open 2015 February; 4 (2): 180-185



(2) 効率上昇

oocyte injection & host transfer 法


K. Nakajima and Y. Yaoita. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis. Biology Open 2015 February; 4 (2): 180-185




oocyte injection & intracytoplasmic sperm injection (ICSI) 法


K. Miyamoto, K. T. Suzuki, M. Suzuki, Y. Sakane,
T. Sakuma, S. Herberg, A. Simeone, D. Simpson, J. Jullien, T. Yamamoto, J. B. Gurdon. The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos. PLOS ONE 2015 November; 18




(3) 組織特異(優先)的発現

生殖細胞:ノックアウト致死遺伝子のノックアウトホモ個体を得る事が可能に


K. Nakajima and Y. Yaoita. Development of a new approach for targeted gene editing in primordial germ cells using TALENs in Xenopus. Biology Open 2015 March; 4(3): 259-266




(4) ノックイン

TAL-PITCh


S. Nakade, T. Tsubota, Y. Sakane, S. Kume, N. Sakamoto, M. Obara, T. Daimon, H. Sezutsu, T. Yamamoto, T.Sakuma and K. T. Suzuki. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nature COMMUNICATIONS 2014; 5: 5560





更新:2018.9.25