NBRP_Clawed_frogs_Newts_Top_EN.html

Experimental Protocols

  1. •Artificial insemination method

  2. Practically demonstrated at the 10th NIBB International Practical Course Genome Editing and Imaging of Fish and Amphibians, which was held at the National Institute for Basic Biology, from 20-29th September, 2018.

Artificial insemination (from testes)

Artificial insemination (collecting eggs and inseminating)

Artificial insemination (de-jelly)

  1. Japanese / English

References

Papers that published useful protocols for users are introduced here. These include the protocols for the gene editing techniques that were developed using NBRP X. tropicalis. This also includes papers in which the frogs provided by this resource center were used for experiments.


The protocols, developed by Xenopus tropicalis NBR Project.
The papers, in which X. tropicalis resources were provided by the NBRP.
The papers, in which X. tropicalis resources were provided by other facilities and projects.




CRISPR/Cas9

(1) Basic techniques

sgRNA construction using PCR, etc.


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) High-efficiency KO

Production of F0 heterozygous KO embryos without chimera using oocyte injection and host transfer


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) Germ cell specific KO (Leapfrogging)

Production of KO embryos, of which gene disruption is restricted to germ cells, and then the next generation produces lethal embryos homozygous for the disrupted gene



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



(4) Kidney specific KO


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



(5) Knock-in

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



(6) その他

Base modification


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) Basic techniques

Original methods for TALEN construction

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




Key points for making TALEN construction


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




Optimal TALEN vector selection from various modified vectors using X. tropicalis


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




Time course of TALEN KO protocol


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) High-efficiency TALEN

The oocyte injection and host transfer method


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




The oocyte injection and intracytoplasmic sperm injection method


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) Germ cell specific KO

It is available to produce KO embryos, of which disrupted allele homozygosity is lethal in early development.


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) Knock-in

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



9.25.2018 update


 

  1. •Preservation of isolated testes of X. tropicalis (10.30.2019 update)

Isolated testes of X. tropicalis can be preserved for after removal from the male body. We confirmed that healthy testes (90 % fertilization rate immediately after removal) retain 45% average fertilization rate when stored in 190% Steinberg + gentamicin solution (see Figure below) at 14 ° C for 3 days (see Table below).

We are currently verifying the fertilization rate when stored at a temperature other than 14 ℃.

Table. Preservation conditions and fertilization rates

Condition

0 day after removal

1

3 days after removal

Male No.

Strain

Soaking

solution

Pres-ervation

(℃)

Total

eggs

Fertilized

eggs

Fer-tilization

rate (%)

Total

eggs

Fertilized

eggs

Fer-tilization

rate (%)

2

3

Nigerian A

Nigerian A

Nigerian A

190%

steinberg

+ gentamicin

190%

steinberg

+ gentamicin

190%

steinberg

+ gentamicin

14

14

22

584

710

no data

524

642

no data

89.7

90.4

no data

510

350

709

301

106

490

60.1

30.3

69.1

Figure. 190% Steinberg solution (pH 7.8〜8.0)

Methods

  1. •RNA microinjection into eggs of X. tropicalis (4.19.2022 update)

    Pleas read this PDF document.

Released Videos

  1. •Microinjection (4.20.2022 update)

  1. •Preparation of capillaries for microinjection into eggs of X. tropicalis (4.20.2022 update)

  1. •Oocyte maturation of X. tropicalis (4.20.2022 update)

Microinjection into the two-cell stage eggs of Xenopus tropicalis

  1. •Sperm cryopreservation method for Xenopus tropicalis (5.27.2022.update)

Freezing

Fertilization

  1. Experimental Protocols