Animal Reproduction (AR)
Animal Reproduction (AR)
Thematic Section: 34th Annual Meeting of the Brazilian Embryo Technology Society (SBTE)

Light up the embryos: knock-in reporter generation for mouse developmental biology

Bin Gu

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Abstract: Developmental biology seeks to understand the sophisticated regulated process through which a single cell – a fertilized egg – generates a highly organized organism. The most effective way to reveal the nature of these processes is to follow single cells and cell lineages in real-time. Recent advances in imaging equipment, fluorescent tags and computational tools have made long term multi-color imaging of cells and embryos possible. However, there is still one major challenging for achieving live imaging of mammalian embryos- the generation of embryos carrying reporters that recapitulate the endogenous expression pattern of marker genes. Recent developments of genome editing technology played important roles in enabling efficient generation of reporter mouse models. This mini review discusses recent developments of technologies for efficiently generate knock-in reporter mice and the application of these models in live imaging development. With these developments, we are starting to realize the long-sought promises of realtime analysis of mammalian development.


CRISPR-Cas9, genome editing, knock-in reporter, HDR, embryo


Amat F, Hockendorf B, Wan Y, Lemon WC, McDole K, Keller PJ. Efficient processing and analysis of large-scale light-sheet microscopy data. Nat Protoc. 2015;10(11):1679-96. PMid:26426501.

Benninger RK, Hao M, Piston DW. Multi-photon excitation imaging of dynamic processes in living cells and tissues. Rev Physiol Biochem Pharmacol. 2008;160:71-92. PMid:18418560.

Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, Aumonier S, Gotthard G, Royant A, Hink MA, Gadella TW Jr. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods. 2017;14(1):53-6. PMid:27869816.

Capecchi MR. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet. 2005;6(6):507-12. PMid:15931173.

Chen S, Sun S, Moonen D, Lee C, Lee AY, Schaffer DV, He L. CRISPR-READI: efficient generation of knockin mice by CRISPR RNP electroporation and AAV Donor infection. Cell Rep. 2019;27(13):3780-9.e4.

Codner GF, Mianne J, Caulder A, Loeffler J, Fell R, King R, Allan AJ, Mackenzie M, Pike FJ, McCabe CV, Christou S, Joynson S, Hutchison M, Stewart ME, Kumar S, Simon MM, Agius L, Anstee QM, Volynski KE, Kullmann DM, Wells S, Teboul L. Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol. 2018;16(1):70. PMid:29925374.

Cohen J. ‘Any idiot can do it.’ Genome editor CRISPR could put mutant mice in everyone’s reach. Science. 2016:89407621.

Goodwin LO, Splinter E, Davis TL, Urban R, He H, Braun RE, Chesler EJ, Kumar V, van Min M, Ndukum J, Philip VM, Reinholdt LG, Svenson K, White JK, Sasner M, Lutz C, Murray SA. Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome Res. 2019;29(3):494-505. PMid:30659012.

Gu B, Posfai E, Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol. 2018;36(7):632-7. PMid:29889212.

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-78. PMid:24906146.

Hustedt N, Durocher D. The control of DNA repair by the cell cycle. Nat Cell Biol. 2016;19(1):1-9. PMid:28008184.

Ikawa M, Yamada S, Nakanishi T, Okabe M. ‘Green mice’ and their potential usage in biological research. FEBS Lett. 1998;430(1-2):83-7. PMid:9678599.

Ittner LM, Gotz J. Pronuclear injection for the production of transgenic mice. Nat Protoc. 2007;2(5):1206-15. PMid:17546016.

Kolberg K, Puettmann C, Pardo A, Fitting J, Barth S. SNAP-tag technology: a general introduction. Curr Pharm Des. 2013;19(30):5406-13. PMid:23431982.

Laboulaye MA, Duan X, Qiao M, Whitney IE, Sanes JR. Mapping Transgene Insertion Sites Reveals Complex Interactions Between Mouse Transgenes and Neighboring Endogenous Genes. Front Mol Neurosci. 2018;11:385. PMid:30405348.

Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 2014;3:e04766. PMid:25497837.

Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M, Simpson D, Mendez J, Zimmerman K, Otto P, Vidugiris G, Zhu J, Darzins A, Klaubert DH, Bulleit RF, Wood KV. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol. 2008;3(6):373-82. PMid:18533659.

Ma M, Zhuang F, Hu X, Wang B, Wen XZ, Ji JF, Xi JJ. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res. 2017;27(4):578-81. PMid:28266543.

McDole K, Guignard L, Amat F, Berger A, Malandain G, Royer LA, Turaga SC, Branson K, Keller PJ. In Toto Imaging and reconstruction of post-implantation mouse development at the single-cell level. Cell. 2018;175(3):859-76.e33.

Miura H, Quadros RM, Gurumurthy CB, Ohtsuka M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc. 2018;13(1):195-215. PMid:29266098.

Mizuno N, Mizutani E, Sato H, Kasai M, Ogawa A, Suchy F, Yamaguchi T, Nakauchi H. Intra-embryo gene cassette knockin by CRISPR/Cas9-mediated genome editing with adeno-associated viral vector. iScience. 2018;9:286-97.

Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki KT. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun. 2014;5(1):5560. PMid:25410609.

Palmiter RD, Norstedt G, Gelinas RE, Hammer RE, Brinster RL. Metallothionein-human GH fusion genes stimulate growth of mice. Science. 1983;222(4625):809-14. PMid:6356363.

Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D, Buckley SM, Seshacharyulu P, Batra SK, Behlke MA, Zeiner SA, Jacobi AM, Izu Y, Thoreson WB, Urness LD, Mansour SL, Ohtsuka M, Gurumurthy CB. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 2017;18(1):92. PMid:28511701.

Rodriguez AJ, Condeelis J, Singer RH, Dictenberg JB. Imaging mRNA movement from transcription sites to translation sites. Semin Cell Dev Biol. 2007;18(2):202-8. PMid:17376719.

Shcherbakova DM, Baloban M, Verkhusha VV. Near-infrared fluorescent proteins engineered from bacterial phytochromes. Curr Opin Chem Biol. 2015;27:52-63. PMid:26115447.

Svoboda P. Mammalian zygotic genome activation. Semin Cell Dev Biol. 2018;84:118-26. PMid:29233752.

Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159(3):635-46. PMid:25307933.

Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51(3):503-12. PMid:2822260.

Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67(1):509-44. PMid:9759496.

Wan Y, McDole K, Keller PJ. Light-sheet microscopy and its potential for understanding developmental processes. Annu Rev Cell Dev Biol. 2019;35(1):655-81. PMid:31299171.

Weis J, Fine SM, Sanes JR. Integration site-dependent transgene expression used to mark subpopulations of cells in vivo: an example from the neuromuscular junction. Brain Pathol. 1992;2(1):31-7. PMid:1341945.

Yang D, Scavuzzo MA, Chmielowiec J, Sharp R, Bajic A, Borowiak M. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep. 2016;6(1):21264. PMid:26887909.

Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154(6):1370-9. PMid:23992847.

Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang P, Sun Q, Shi L, Yang H. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017;27(6):801-14. PMid:28524166.

Yoon Y, Wang D, Tai PWL, Riley J, Gao G, Rivera-Perez JA. Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses. Nat Commun. 2018;9(1):412. PMid:29379011.

Zhu M, Tao H, Samani M, Luo M, Wang X, Hopyan S, Sun Y. Spatial mapping of tissue properties in vivo reveals a 3D stiffness gradient in the mouse limb bud. Proc Natl Acad Sci USA. 2020;117(9):4781-91. PMid:32071242.

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