Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    CAS  Article  Google Scholar 

  2. 2

    Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  Article  Google Scholar 

  3. 3

    Gaj, T., Gersbach, C.A. & Barbas, C.F. III. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).

    CAS  Article  Google Scholar 

  4. 4

    Geurts, A.M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Sung, Y.H. et al. Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 23–24 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  Article  Google Scholar 

  7. 7

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  Article  Google Scholar 

  8. 8

    Sunagawa, G.A. et al. Mammalian reverse genetics without crossing reveals Nr3a as a short-sleeper gene. Cell Rep. 14, 662–677 (2016).

    CAS  Article  Google Scholar 

  9. 9

    Tatsuki, F. et al. Involvement of Ca(2+)-dependent hyperpolarization in sleep duration in mammals. Neuron 90, 70–85 (2016).

    CAS  Article  Google Scholar 

  10. 10

    Yang, H., Wang, H. & Jaenisch, R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat. Protoc. 9, 1956–1968 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).

    CAS  Article  Google Scholar 

  12. 12

    Nakao, H. et al. A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes. Genesis 54, 65–77 (2016).

    CAS  Article  Google Scholar 

  13. 13

    Menoret, S. et al. Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins. Sci. Rep. 5, 14410 (2015).

    CAS  Article  Google Scholar 

  14. 14

    Aida, T. et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol. 16, 87 (2015).

    Article  Google Scholar 

  15. 15

    Wang, L. et al. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos. Sci. Rep. 5, 17517 (2015).

    CAS  Article  Google Scholar 

  16. 16

    Yoshimi, K. et al. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat. Commun. 7, 10431 (2016).

    CAS  Article  Google Scholar 

  17. 17

    Nagy, A. et al. Embryonic stem cells alone are able to support fetal development in the mouse. Development 110, 815–821 (1990).

    CAS  PubMed  Google Scholar 

  18. 18

    Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J.C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428 (1993).

    CAS  Article  Google Scholar 

  19. 19

    Wang, Z.Q., Kiefer, F., Urbanek, P. & Wagner, E.F. Generation of completely embryonic stem cell-derived mutant mice using tetraploid blastocyst injection. Mech. Dev. 62, 137–145 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Schwenk, F. et al. Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological, physiological, and neurological characteristics. Mol. Cell Biol. 23, 3982–3989 (2003).

    CAS  Article  Google Scholar 

  21. 21

    George, S.H. et al. Developmental and adult phenotyping directly from mutant embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 4455–4460 (2007).

    CAS  Article  Google Scholar 

  22. 22

    Seibler, J. et al. Rapid generation of inducible mouse mutants. Nucleic Acids Res. 31, e12 (2003).

    Article  Google Scholar 

  23. 23

    Li, X. et al. The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation. Reproduction 130, 53–59 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Eakin, G.S., Hadjantonakis, A.K., Papaioannou, V.E. & Behringer, R.R. Developmental potential and behavior of tetraploid cells in the mouse embryo. Dev. Biol. 288, 150–159 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Lu, T.Y. & Markert, C.L. Manufacture of diploid/tetraploid chimeric mice. Proc. Natl. Acad. Sci. USA 77, 6012–6016 (1980).

    CAS  Article  Google Scholar 

  26. 26

    Eggan, K. et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. USA 98, 6209–6214 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Gertsenstein, M. et al. Efficient generation of germ line transmitting chimeras from C57BL/6N ES cells by aggregation with outbred host embryos. PLoS One 5, e11260 (2010).

    Article  Google Scholar 

  28. 28

    Poueymirou, W.T. et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat. Biotechnol. 25, 91–99 (2007).

    CAS  Article  Google Scholar 

  29. 29

    Huang, J. et al. Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation. Stem Cells 26, 1883–1890 (2008).

    CAS  Article  Google Scholar 

  30. 30

    Johnson, M.H. & McConnell, J.M. Lineage allocation and cell polarity during mouse embryogenesis. Semin. Cell Dev. Biol. 15, 583–597 (2004).

    CAS  Article  Google Scholar 

  31. 31

    Johnson, M.H., Maro, B. & Takeichi, M. The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres. J. Embryol. Exp. Morph. 93, 239–255 (1986).

    CAS  PubMed  Google Scholar 

  32. 32

    Kiyonari, H., Kaneko, M., Abe, S. & Aizawa, S. Three inhibitors of FGF receptor, ERK, and GSK3 establishes germline-competent embryonic stem cells of C57BL/6N mouse strain with high efficiency and stability. Genesis 48, 317–327 (2010).

    CAS  PubMed  Google Scholar 

  33. 33

    Susaki, E.A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    CAS  Article  Google Scholar 

  34. 34

    Tainaka, K. et al. Whole-body imaging with single-cell resolution by tissue decolorization. Cell 159, 911–924 (2014).

    CAS  Article  Google Scholar 

  35. 35

    Wang, Y. et al. Highly efficient generation of biallelic reporter gene knock-in mice via CRISPR-mediated genome editing of ESCs. Protein Cell 7, 152–156 (2016).

    Article  Google Scholar 

  36. 36

    Nagy, A., Nagy, K. & Gertsenstein, M. Production of mouse chimeras by aggregating pluripotent stem cells with embryos. Methods Enzymol. 476, 123–149 (2010).

    CAS  Article  Google Scholar 

  37. 37

    Ode, K.L. et al. Knockout-rescue embryonic stem cell-derived mouse reveals circadian-period control by quality and quantity of CRY1. Mol. Cell 65, 176–190 (2017).

    CAS  Article  Google Scholar 

  38. 38

    Sato, H., Amagai, K., Shimizukawa, R. & Tamai, Y. Stable generation of serum- and feeder-free embryonic stem cell-derived mice with full germline-competency by using a GSK3 specific inhibitor. Genesis 47, 414–422 (2009).

    CAS  Article  Google Scholar 

  39. 39

    Sommer, D., Peters, A.E., Baumgart, A.K. & Beyer, M. TALEN-mediated genome engineering to generate targeted mice. Chromosome Res. 23, 43–55 (2015).

    CAS  Article  Google Scholar 

  40. 40

    Vanamee, E.S., Santagata, S. & Aggarwal, A.K. FokI requires two specific DNA sites for cleavage. J. Mol. Biol. 309, 69–78 (2001).

    CAS  Article  Google Scholar 

  41. 41

    Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).

    CAS  Article  Google Scholar 

  42. 42

    Hermann, M., Cermak, T., Voytas, D.F. & Pelczar, P. Mouse genome engineering using designer nucleases. J. Vis. Exp. 86, e50930 (2014).

    Google Scholar 

  43. 43

    Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

    CAS  Article  Google Scholar 

  44. 44

    Doyle, E.L. et al. TAL effector-nucleotide targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 40, W117–W122 (2012).

    CAS  Article  Google Scholar 

  45. 45

    Hale, C.R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009).

    CAS  Article  Google Scholar 

  46. 46

    Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

    CAS  Article  Google Scholar 

  47. 47

    Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  Article  Google Scholar 

  48. 48

    Shen, B. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11, 399–402 (2014).

    CAS  Article  Google Scholar 

  49. 49

    Kleinstiver, B.P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  Google Scholar 

  50. 50

    Hirano, S., Nishimasu, H., Ishitani, R. & Nureki, O. Structural basis for the altered PAM specificities of engineered CRISPR-Cas9. Mol. Cell 61, 886–894 (2016).

    CAS  Article  Google Scholar 

  51. 51

    Bae, S., Kweon, J., Kim, H.S. & Kim, J.S. Microhomology-based choice of Cas9 nuclease target sites. Nat. Methods 11, 705–706 (2014).

    CAS  Article  Google Scholar 

  52. 52

    Li, H.L. et al. Precise correction of the dystrophin gene in Duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Rep. 4, 143–154 (2015).

    CAS  Article  Google Scholar 

  53. 53

    Nakade, S. et al. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat. Commun. 5, 5560 (2014).

    CAS  Article  Google Scholar 

  54. 54

    Sakuma, T. et al. Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids. Int. J. Mol. Sci. 16, 23849–23866 (2015).

    CAS  Article  Google Scholar 

  55. 55

    Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K.T. & Yamamoto, T. MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat. Protoc. 11, 118–133 (2016).

    CAS  Article  Google Scholar 

  56. 56

    Murata, T. et al. ang is a novel gene expressed in early neuroectoderm, but its null mutant exhibits no obvious phenotype. Gene Exp. Patterns 5, 171–178 (2004).

    CAS  Article  Google Scholar 

  57. 57

    Giraldo, P. & Montoliu, L. Size matters: use of YACs, BACs and PACs in transgenic animals. Transg. Res. 10, 83–103 (2001).

    CAS  Article  Google Scholar 

  58. 58

    Qi, X. et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc. Natl. Acad. Sci. USA 101, 6027–6032 (2004).

    CAS  Article  Google Scholar 

  59. 59

    Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254–1269 (2014).

    CAS  Article  Google Scholar 

  60. 60

    Dutta, D. et al. Self-renewal versus lineage commitment of embryonic stem cells: protein kinase C signaling shifts the balance. Stem Cells 29, 618–628 (2011).

    CAS  Article  Google Scholar 

  61. 61

    Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    CAS  Article  Google Scholar 

  62. 62

    Zhang, P., Wu, X., Hu, C., Wang, P. & Li, X. Rho kinase inhibitor Y-27632 and accutase dramatically increase mouse embryonic stem cell derivation. In Vitro Cell. Dev. Biol. Anim. 48, 30–36 (2012).

    Article  Google Scholar 

  63. 63

    Lee, J.H., Hart, S.R. & Skalnik, D.G. Histone deacetylase activity is required for embryonic stem cell differentiation. Genesis 38, 32–38 (2004).

    CAS  Article  Google Scholar 

  64. 64

    Ware, C.B. et al. Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells. Cell Stem Cell 4, 359–369 (2009).

    CAS  Article  Google Scholar 

  65. 65

    Hezroni, H., Sailaja, B.S. & Meshorer, E. Pluripotency-related, valproic acid (VPA)-induced genome-wide histone H3 lysine 9 (H3K9) acetylation patterns in embryonic stem cells. J. Biol. Chem. 286, 35977–35988 (2011).

    CAS  Article  Google Scholar 

  66. 66

    Blaschke, K. et al. Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500, 222–226 (2013).

    CAS  Article  Google Scholar 

  67. 67

    Nakao, K., Nakagata, N & Katsuki, M. Simple and efficient vitrification procedure for cryopreservation of mouse embryos. Exp. Anim. 46, 231–234 (1997).

    CAS  Article  Google Scholar 

  68. 68

    Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    CAS  Article  Google Scholar 

  69. 69

    Abe, T. et al. Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging. Genesis 49, 579–590 (2011).

    CAS  Article  Google Scholar 

  70. 70

    Hohenstein, P. et al. High-efficiency Rosa26 knock-in vector construction for Cre-regulated overexpression and RNAi. PathoGenetics 1, 3 (2008).

    Article  Google Scholar 

Download references


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.).

Author information




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.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing