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Production of knock-in mice in a single generation from embryonic stem cells

Abstract

The system-level identification and analysis of molecular networks in mammals can be accelerated by 'next-generation' genetics, defined as genetics that does not require crossing of multiple generations of animals in order to achieve the desired genetic makeup. We have established a highly efficient procedure for producing knock-in (KI) mice within a single generation, by optimizing the genome-editing protocol for KI embryonic stem (ES) cells and the protocol for the generation of fully ES-cell-derived mice (ES mice). Using this protocol, the production of chimeric mice is eliminated, and, therefore, there is no requirement for the crossing of chimeric mice to produce mice that carry the KI gene in all cells of the body. Our procedure thus shortens the time required to produce KI ES mice from about a year to 3 months. Various kinds of KI ES mice can be produced with a minimized amount of work, facilitating the elucidation of organism-level phenomena using a systems biology approach. In this report, we describe the basic technologies and protocols for this procedure, and discuss the current challenges for next-generation mammalian genetics in organism-level systems biology studies.

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Figure 1: Overview of advanced ES-mouse protocol.
Figure 2: Simple and efficient protocols for establishing KI ESCs under feeder-free conditions.
Figure 3: Screening and genotyping procedures for KI ESCs, and typical results.
Figure 4: Injection of KI ESCs into 8-cell-stage embryos to produce KI ES mice.

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Acknowledgements

We thank our laboratory members at RIKEN QBiC, RIKEN CLST, and the University of Tokyo, in particular, S. Morino and M. Muramatsu, for their help in maintaining the ESCs and preparing materials; J. Garçon and K. Yamanaka for producing ES mice; M. Ukai for help with the genotyping of ESCs; and M. Kaneko, M. Shigeta and M. Okugawa for help in preparing the manuscript. This work was supported by grants from AMED-CREST (H.R.U.), CREST (H.R.U.), Brain/MINDS (H.R.U.), the Basic Science and Platform Technology Program for Innovative Biological Medicine (H.R.U.), and the Cell Innovation Program (H.R.U.), KAKENHI Grants-in-Aid from JSPS (Scientific Research S, 25221004, H.R.U.; Scientific Research on Innovative Areas, 23115006, H.R.U.), and the strategic programs for R&D of RIKEN (H.R.U.), an intramural Grant-in-Aid from the RIKEN QBiC (H.R.U.), and grants from Takeda Science Foundation (H.R.U.).

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Authors

Contributions

H.R.U., H.U. and H.K. designed the study and wrote the manuscript. H.U. developed, improved, and performed most of the protocols related to knock-in ESC establishment. H.K. developed most of the 8-cell injection protocol. All authors discussed the results and commented on the manuscript text.

Corresponding author

Correspondence to Hiroki R Ueda.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Number of pups and chimeras. (PDF 109 kb)

Collection of ESCs.

A video showing how to fill an injection pipette with ES cells. Move the injection pipette to the ESC drop and collect as many ESCs as needed to inject in one batch. If the pipette becomes clogged, push the ES cells out once and collect again. (MP4 29103 kb)

Injection into 8-cell-stage embryos.

A video showing how ES cells are injected into 8-cell-stage embryos. Hold the 8-cell-stage embryo with a holding pipette and insert the injection pipette filled with ES cells into the embryo. Then, inject 10–30 ESCs into each embryo to fill the perivitelline space. (MP4 3764 kb)

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Ukai, H., Kiyonari, H. & Ueda, H. Production of knock-in mice in a single generation from embryonic stem cells. Nat Protoc 12, 2513–2530 (2017). https://doi.org/10.1038/nprot.2017.110

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