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In Ovo Electroporation of miRNA Plasmids to Silence Genes in a Temporally and Spatially Controlled Manner

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MicroRNA Technologies

Part of the book series: Neuromethods ((NM,volume 128))

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Abstract

The ability to spatially and temporally control gene expression during development is crucial for the elucidation of gene function in vivo. The use of RNA interference (RNAi)-based technologies in combination with oviparous animal models allows for efficient, precise gene silencing. We have developed approaches using RNAi in the chicken embryo to analyze gene function during neural tube development. Here we describe the construction of plasmids that direct the expression of one or two artificial microRNAs (miRNAs) to knock down endogenous target protein/s upon electroporation into the spinal cord. The miRNA cassette is directly linked to a fluorescent protein reporter, which allows the faithful visualization of transfected cells. Different promoters/enhancers drive transcript expression in genetically defined cell subpopulations in the neural tube. Mixing multiple RNAi vectors allows combinatorial knockdowns of two or more genes in different cell types, thus permitting the rapid analysis of complex cellular and molecular interactions.

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References

  1. Friedel RH, Wurst W, Wefers B, Kühn R (2011) Generating conditional knockout mice. Methods Mol Biol 693:205–231

    Article  CAS  PubMed  Google Scholar 

  2. Pekarik V, Bourikas D, Miglino N, Joset P, Preiswerk S, Stoeckli ET (2003) Screening for gene function in chicken embryo using RNAi and electroporation. Nat Biotechnol 21:93–96

    Article  CAS  PubMed  Google Scholar 

  3. Bourikas D, Pekarik V, Baeriswyl T, Grunditz A, Sadhu R, Nardó M, Stoeckli ET (2005) Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat Neurosci 8:297–304

    Article  CAS  PubMed  Google Scholar 

  4. Mauti O, Domanitskaya E, Andermatt I, Sadhu R, Stoeckli ET (2007) Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system. Neural Dev 2:28

    Article  PubMed  PubMed Central  Google Scholar 

  5. Baeriswyl T, Stoeckli ET (2008) Axonin-1/TAG-1 is required for pathfinding of granule cell axons in the developing cerebellum. Neural Dev 3:7

    Article  PubMed  PubMed Central  Google Scholar 

  6. Niederkofler V, Baeriswyl T, Ott R, Stoeckli ET (2010) Nectin-like molecules/SynCAMs are required for post-crossing commissural axon guidance. Development 137:427–435

    Article  CAS  PubMed  Google Scholar 

  7. Domanitskaya E, Wacker A, Mauti O, Baeriswyl T, Esteve P, Bovolenta P, Stoeckli ET (2010) Sonic hedgehog guides post-crossing commissural axons both directly and indirectly by regulating Wnt activity. J Neurosci 30:11167–11176

    Article  CAS  PubMed  Google Scholar 

  8. Katahira T, Nakamura H (2003) Gene silencing in chick embryos with a vector-based small interfering RNA system. Dev Growth Differ 45:361–367

    Article  CAS  PubMed  Google Scholar 

  9. Das RM, van Hateren NJ, Howell GR, Farrell ER, Bangs FK, Porteous VC, Manning EM, McGrew MJ, Ohyama K, Sacco MA et al (2006) A robust system for RNA interference in the chicken using a modified microRNA operon. Dev Biol 294:554–563

    Article  CAS  PubMed  Google Scholar 

  10. Kurreck J (2009) RNA interference: from basic research to therapeutic applications. Angew Chem Int Ed Engl 48:1378–1398

    Article  CAS  PubMed  Google Scholar 

  11. Wilson NH, Stoeckli ET (2011) Cell type specific, traceable gene silencing for functional gene analysis during vertebrate neural development. Nucleic Acids Res 39(20):e133. doi:10.1093/nar/gkr628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lee S-K, Jurata LW, Funahashi J, Ruiz EC, Pfaff SL (2004) Analysis of embryonic motoneuron gene regulation: derepression of general activators function in concert with enhancer factors. Development 131:3295–3306

    Article  CAS  PubMed  Google Scholar 

  13. Uemura O, Okada Y, Ando H, Guedj M, Higashijima S-I, Shimazaki T, Chino N, Okano H, Okamoto H (2005) Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression. Dev Biol 278:587–606

    Article  CAS  PubMed  Google Scholar 

  14. Avraham O, Hadas Y, Vald L, Zisman S, Schejter A, Visel A, Klar A (2009) Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1. Neural Dev 4:21

    Article  PubMed  PubMed Central  Google Scholar 

  15. Timmer J, Johnson J, Niswander L (2001) The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers. Genesis 29:123–132

    Article  CAS  PubMed  Google Scholar 

  16. Kieleczawa J (2006) Fundamentals of sequencing of difficult templates—an overview. J Biomol Tech 17:207–217

    PubMed  PubMed Central  Google Scholar 

  17. Wilson NH, Stoeckli ET (2012) In ovo electroporation of miRNA-based plasmids in the developing neural tube and assessment of phenotypes by DiI injection in open-book preparations. J Vis Exp 68:e4384. doi:10.3791/4384

    Google Scholar 

  18. Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195:231–272

    Article  CAS  PubMed  Google Scholar 

  19. Baeriswyl T, Mauti O, Stoeckli ET (2008) Temporal control of gene silencing by in ovo electroporation. Methods Mol Biol 442:231–244

    Article  CAS  PubMed  Google Scholar 

  20. Cullen BR (2006) Enhancing and confirming the specificity of RNAi experiments. Nat Methods 3:677–681

    Article  CAS  PubMed  Google Scholar 

  21. Krull CE (2004) A primer on using in ovo electroporation to analyze gene function. Dev Dyn 229:433–439

    Article  CAS  PubMed  Google Scholar 

  22. Croteau LP, Kania A (2011) Optimisation of in ovo electroporation of the chick neural tube. J Neurosci Methods 201(2):381–384. doi:10.1016/j.jneumeth.2011.08.012

    Article  CAS  PubMed  Google Scholar 

  23. Rao M, Baraban JH, Rajaii F, Sockanathan S (2004) In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo. Dev Dyn 231:592–600

    Article  CAS  PubMed  Google Scholar 

  24. Stepanek L, Stoker AW, Stoeckli E, Bixby JL (2005) Receptor tyrosine phosphatases guide vertebrate motor axons during development. J Neurosci 25:3813–3823

    Article  CAS  PubMed  Google Scholar 

  25. Wilson NH, Stoeckli ET (2013) Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner. Neuron 79(3):478–491. doi:10.1016/j.neuron.2013.05.025

    Article  CAS  PubMed  Google Scholar 

  26. Sato F, Nakagawa T, Ito M, Kitagawa Y, Hattori M-A (2004) Application of RNA interference to chicken embryos using small interfering RNA. J Exp Zool A Comp Exp Biol 301:820–827

    Article  PubMed  Google Scholar 

  27. Andermatt I, Wilson NH, Bergmann T, Mauti O, Gesemann M, Sockanathan S, Stoeckli ET (2014) Semaphorin 6B acts as a receptor in post-crossing commissural axon guidance. Development 141(19):3709–3720. doi:10.1242/dev.112185

    Article  CAS  PubMed  Google Scholar 

  28. Chesnutt C, Niswander L (2004) Plasmid-based short-hairpin RNA interference in the chicken embryo. Genesis 39:73–78

    Article  CAS  PubMed  Google Scholar 

  29. Perrin FE, Rathjen FG, Stoeckli ET (2001) Distinct subpopulations of sensory afferents require F11 or axonin-1 for growth to their target layers within the spinal cord of the chick. Neuron 30(3):707–723

    Article  CAS  PubMed  Google Scholar 

  30. Frei JA, Andermatt I, Gesemann M, Stoeckli ET (2014) The SynCAM synaptic cell adhesion molecules are involved in sensory axon pathfinding by regulating axon-axon contacts. J Cell Sci 127:5288–5302. doi:10.1242/jcs.157032

    Article  PubMed  Google Scholar 

  31. Perrin FE, Stoeckli ET (2000) Use of lipophilic dyes in studies of axonal pathfinding in vivo. Microsc Res Tech 48:25–31

    Article  CAS  PubMed  Google Scholar 

  32. Wilson NH, Stoeckli ET (2014) Open-book preparations from chick embryos and DiI labeling of commissural axons. Bio-Protocol 4(13):e1176, http://www.bio-protocol.org/e1176

    Article  Google Scholar 

  33. Witkos TM, Koscianska E, Krzyzosiak WJ (2011) Practical aspects of microRNA target prediction. Curr Mol Med 11(2):93–109. doi:10.2174/156652411794859250

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ehlert EM, Eggers R, Niclou SP, Verhaagen J (2010) Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system. BMC Neurosci 11:20

    Article  PubMed  PubMed Central  Google Scholar 

  35. Wilczynska A, Bushell M (2015) The complexity of miRNA-mediated repression. Cell Death Differ 22(1):22–33. doi:10.1038/cdd.2014.112

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Queensland Brain Institute, University of Queensland, Brisbane, 4072, Australia

    Nicole H. Wilson

  2. Institute of Molecular Life Sciences and Neuroscience Center Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Esther T. Stoeckli

Authors
  1. Nicole H. Wilson
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  2. Esther T. Stoeckli
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Corresponding author

Correspondence to Esther T. Stoeckli .

Editor information

Editors and Affiliations

  1. Institute of Human Genetics, University of Cologne, Cologne, Germany

    Min Jeong Kye

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© 2016 Springer Science+Business Media New York

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Wilson, N.H., Stoeckli, E.T. (2016). In Ovo Electroporation of miRNA Plasmids to Silence Genes in a Temporally and Spatially Controlled Manner. In: Kye, M. (eds) MicroRNA Technologies. Neuromethods, vol 128. Humana Press, New York, NY. https://doi.org/10.1007/7657_2016_5

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  • DOI: https://doi.org/10.1007/7657_2016_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7173-2

  • Online ISBN: 978-1-4939-7175-6

  • eBook Packages: Springer Protocols

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