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Direct analysis of brain phenotypes via neural blastocyst complementation

Nature Protocols volume 15, pages 3154–3181 (2020)Cite this article

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Abstract

We provide a protocol for generating forebrain structures in vivo from mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC). We developed this protocol for studies of development and function of specific forebrain regions, including the cerebral cortex and hippocampus. We describe a complete workflow, from methods for modifying a given genomic locus in ESCs via CRISPR–Cas9-mediated editing to the generation of mouse chimeras with ESC-reconstituted forebrain regions that can be directly analyzed. The procedure begins with genetic editing of mouse ESCs via CRISPR–Cas9, which can be accomplished in ~4–8 weeks. We provide protocols to achieve fluorescent labeling of ESCs in ~2–3 weeks, which allows tracing of the injected, ESC-derived donor cells in chimeras generated via NBC. Once modified ESCs are ready, NBC chimeras are generated in ~3 weeks via injection of ESCs into genetically programmed blastocysts that are subsequently transferred into pseudo-pregnant fosters. Our in vivo brain organogenesis platform is efficient, allowing functional and systematic analysis of genes and other genomic factors in as little as 3 months, in the context of a whole organism.

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Fig. 1: Overview of mouse early embryonic development and the NBC organogenesis approach.
Fig. 2: Schematic of the breeding crosses used to generate DsRed-labeled NBC host blastocysts.
Fig. 3: Lentiviral transduction of ESCs.
Fig. 4: ESC clone picking.
Fig. 5: DNA isolation from ESC clones and karyotyping.
Fig. 6: Collection of blastocysts.
Fig. 7: Transfer pipette setup for blastocyst collection and uterine transfer surgery of injected blastocysts and microinjection holding needle.
Fig. 8: Microinjection needle, ESC injection and overview of microinjection setup.
Fig. 9: PCR genotyping of NBC chimeras and representative photograph of newborn mice.

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Data availability

All data referred to or analyzed are included in Chang et al., Nature (2018): https://doi.org/10.1038/s41586-018-0586-0

References

  1. Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981).

    Article  CAS  Google Scholar 

  2. Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  3. Bradley, A., Evans, M., Kaufman, M. H. & Robertson, E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).

    Article  CAS  Google Scholar 

  4. Miura, T., Mattson, M. P. & Rao, M. S. Cellular lifespan and senescence signaling in embryonic stem cells. Aging Cell 3, 333–343 (2004).

    Article  CAS  Google Scholar 

  5. Bradley, A., Hasty, P., Davis, A. & Ramirez-Solis, R. Modifying the mouse: design and desire. Biotechnology 10, 534–539 (1992).

    CAS  PubMed  Google Scholar 

  6. Chang, A. N. et al. Neural blastocyst complementation enables mouse forebrain organogenesis. Nature 563, 126–130 (2018).

    Article  CAS  Google Scholar 

  7. Kobayashi, T. et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142, 787–799 (2010).

    Article  CAS  Google Scholar 

  8. Chen, J., Lansford, R., Stewart, V., Young, F. & Alt, F. W. RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc. Natl Acad. Sci. USA 90, 4528–4532 (1993).

    Article  CAS  Google Scholar 

  9. Chen, J. et al. Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells. Curr. Biol. 3, 405–413 (1993).

    Article  CAS  Google Scholar 

  10. Usui, J. et al. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am. J. Pathol. 180, 2417–2426 (2012).

    Article  CAS  Google Scholar 

  11. Liégeois, N. J., Horner, J. W. & DePinho, R. A. Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo. Proc. Natl Acad. Sci. USA 93, 1303–1307 (1996).

    Article  Google Scholar 

  12. Stanger, B. Z., Tanaka, A. J. & Melton, D. A. Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver. Nature 445, 886–891 (2007).

    Article  CAS  Google Scholar 

  13. Wu, J. et al. Stem cells and interspecies chimaeras. Nature 540, 51–59 (2016).

    Article  CAS  Google Scholar 

  14. Wu, J. et al. Interspecies chimerism with mammalian pluripotent stem cells. Cell 168, 473–486 (2017).

    Article  CAS  Google Scholar 

  15. Gorski, J. A. et al. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J. Neurosci. 22, 6309–6314 (2002).

    Article  CAS  Google Scholar 

  16. Behringer, R., Gertsenstein, M., Nagy, K. V. & Nagy, A. Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2014).

  17. Conner, D. A. Mouse embryo fibroblast (MEF) feeder cell preparation. Curr. Protoc. Mol. Biol. 23.23.2 (2001).

  18. Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  19. Conner, D. A. Mouse embryonic stem (ES) cell culture. Curr. Protoc. Mol. Biol. 23.23.3 (2001).

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

    Article  CAS  Google Scholar 

  21. Brown, T. Southern blotting. Curr. Protoc. Immunol. 10.10.6A (2001).

  22. Ryder, E. et al. Molecular characterization of mutant mouse strains generated from the EUCOMM/KOMP-CSD ES cell resource. Mamm. Genome 24, 286–294 (2013).

    Article  CAS  Google Scholar 

  23. Kutner, R. H., Zhang, X.-Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495–505 (2009).

    Article  CAS  Google Scholar 

  24. Nissen, S. B. et al. Four simple rules that are sufficient to generate the mammalian blastocyst. PLoS Biol. 15, e2000737 (2017).

    Article  Google Scholar 

  25. Lois, C., Hong, E. J., Pease, S., Brown, E. J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank members of the Alt and Schwer labs and D. Laird for discussions, P.-Y. Huang for help with blastocyst injections and H.-L. Cheng for advice and help with ESC culture. This work was supported by the Howard Hughes Medical Institute, the Boston Children’s Hospital (BCH) Department of Medicine (DOM) Support Fund, the BCH DOM Anderson Porter Fund, a major grant from the Charles H. Hood Foundation (to F.W.A), and a generous gift from the Dabbiere family (to B.S.). B.S. is a Kimmel Scholar of the Sidney Kimmel Foundation and is supported by the University of California, San Francisco (UCSF) Brain Tumor SPORE Career Development Program, the American Cancer Society, the UCSF Program for Breakthrough Biomedical Research (which is partially funded by the Sandler Foundation), the Andrew McDonough B+ Foundation, the Shurl and Kay Curci Foundation and a Martin D. Abeloff V Scholar Award of the V Foundation for Cancer Research. B.S. holds the Suzanne Marie Haderle and Robert Vincent Haderle Endowed Chair at UCSF. H.-Q.D. is a fellow of the Cancer Research Institute of New York. F.W.A. is an Investigator of the Howard Hughes Medical Institute.

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Author notes

  1. These authors contributed equally: Hai-Qiang Dai, Zhuoyi Liang, Amelia N. Chang.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Hai-Qiang Dai, Zhuoyi Liang, Amelia N. Chang, Aimee M. Chapdelaine-Williams & Frederick W. Alt

  2. Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA

    Beatriz Alvarado, Alex A. Pollen & Bjoern Schwer

  3. Department of Neurology, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA, USA

    Alex A. Pollen

  4. Department of Neurological Surgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA, USA

    Bjoern Schwer

Authors
  1. Hai-Qiang Dai
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Contributions

A.N.C., H.-Q.D., Z.L., B.S., and F.W.A. designed the study. A.N.C., H.-Q.D., Z.L., B.A., A.A.P., and B.S. performed experiments. A.M.C.-W. performed NBC injections and related mouse work. B.S. and F.W.A. supervised the research. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Frederick W. Alt or Bjoern Schwer.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Harold Cremer, Lauryl Nutter and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Chang, A. N. et al. Nature 563, 126–130 (2018): https://doi.org/10.1038/s41586-018-0586-0

Key data used in this protocol

Chang, A. N. et al. Nature 563, 126–130 (2018): https://doi.org/10.1038/s41586-018-0586-0

Supplementary information

Reporting Summary

Supplementary Video 1

Dissection of uterine horns.

Supplementary Video 2

Flushing of uterine horns for embryo collection.

Supplementary Video 3

Pulling and blunting the transfer pipette.

Supplementary Video 4

Cutting the microinjection needle after pulling.

Supplementary Video 5

Injection of blastocysts.

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Dai, HQ., Liang, Z., Chang, A.N. et al. Direct analysis of brain phenotypes via neural blastocyst complementation. Nat Protoc 15, 3154–3181 (2020). https://doi.org/10.1038/s41596-020-0364-y

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