Summary
The analysis of gene function during embryonic development asks for tight temporal control of gene expression. Classic genetic tools do not allow for this, as the absence of a gene during the early stages of development will preclude its functional analysis during later stages. In contrast, RNAi technology allows one to achieve temporal control of gene silencing especially when used with oviparous animal models. In contrast to mammals, reptiles and birds are easily accessible during embryonic development. We have developed approaches to use RNAi for the analysis of gene function during nervous system development in the chicken embryo. Although the protocol given here describes a method for gene silencing in the developing spinal cord, it can easily be adapted to other parts of the developing nervous system. The combination of the easy accessibility of the chicken embryo and RNAi provides a unique opportunity for temporal and spatial control of gene silencing during development.
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References
Calegari, F., Marzesco, A. M., Kittler, R., Buchholz, F., and Huttner, W. B. (2004). Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture. Differentiation 72, 92–102
Takahashi, M., Sato, K., Nomura, T., and Osumi, N. (2002). Manipulating gene expressions by electroporation in the developing brain of mammalian embryos. Differentiation 70, 155–162
Bai, J., Ramos, R. L., Ackman, J. B., Thomas, A. M., Lee, R. V., and LoTurco, J. J. (2003). RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat. Neurosci. 6, 1277–1283.
Pekarik, V., Bourikas, D., Miglino, N., Joset, P., Preiswerk, S., and Stoeckli, E. T. (2003). Screening for gene function in chicken embryo using RNAi and electroporation. Nat. Biotechnol. 21, 93–96.
Baeriswyl, T., and Stoeckli, E. T. (2006). In ovo RNAi opens new possibilities for temporal and spatial control of gene silencing during development of the vertebrate nervous system. J. RNAi Gene Silencing 2, 126–135.
Bourikas, D., and Stoeckli, E. T. (2003). New tools for gene manipulation in chicken embryos. Oligonucleotides 13, 411–419.
Prawitt, D., Brixel, L., Spangenberg, C., Eshkind, L., Heck, R., Oesch, F., Zabel, B., and Bockamp, E. (2004). RNAi knock-down mice: An emerging technology for post-genomic functional genetics. Cytogenet. Genome Res. 105, 412–421.
Krull, C. E. (2004). A primer on using in ovo electroporation to analyze gene function. Dev. Dyn. 229, 433–439.
Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., and Yasuda, K. (1999). Efficient targeting of gene expression in chick embryos by microelectroporation. Dev. Growth Differ. 41, 335–344.
Muramatsu, T., Mizutani, Y., Ohmori, Y., and Okumura, J. (1997). Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo. Biochem. Biophys. Res. Commun. 230, 376–380.
Nakamura, H., and Funahashi, J. (2001). Introduction of DNA into chick embryos by in ovo electroporation. Methods 24, 43–48.
Itasaki, N., Bel-Vialar, S., and Krumlauf, R. (1999). “Shocking” developments in chick embryology: Electroporation and in ovo gene expression. Nat. Cell Biol. 1, E203–E207.
Bourikas, D., Pekarik, V., Baeriswyl, T., et al. (2005). Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat. Neurosci. 8, 297–304.
Bron, R., Eickholt, B. J., Vermeren, M., Fragale, N., and Cohen, J. (2004). Functional knockdown of neuropilin-1 in the developing chick nervous system by siRNA hairpins phenocopies genetic ablation in the mouse. Dev. Dyn. 230, 299–308.
Chesnutt, C., and Niswander, L. (2004). Plasmid-based short-hairpin RNA interference in the chicken embryo. Genesis 39, 73–78.
Katahira, T., and Nakamura, H. (2003). Gene silencing in chick embryos with a vector-based small interfering RNA system. Dev. Growth Differ. 45, 361–367.
Toyofuku, T., Zhang, H., Kumanogoh, A., et al. (2004). Guidance of myocardial patterning in cardiac development by Sema6D reverse signalling. Nat. Cell Biol. 6, 1204–1211.
Svoboda, P. (2004). Long dsRNA and silent genes strike back: RNAi in mouse oocytes and early embryos. Cytogenet. Genome Res. 105, 422–434.
Gan, L., Anton, K. E., Masterson, B. A., Vincent, V. A., Ye, S., and Gonzalez-Zulueta, M. (2002). Specific interference with gene expression and gene function mediated by long dsRNA in neural cells. J. Neurosci. Meth. 121, 151–157.
Hillier, L. W., Miller, W., Birney, E., et al. (2004). Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695–716.
Hamburger, V., and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92. Reprinted as Sanes, J. R. (1992). On the republication of the Hamburger–Hamilton stage series. Dev. Dyn. 195, 229–272.
Rao, M., Baraban, J. H., Rajaii, F., and Sockanathan, S. (2004). In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo. Dev. Dyn. 231, 592–600.
Bourikas, D., Baeriswyl, T., Sadhu, R., and Stoeckli, E. T. (2005). In ovo RNAi opens new possibilities for functional genomics in vertebrates. In RNA Interference Technology—From Basic Research to Drug Development (K. Appasani, ed.), Cambridge University Press, Cambridge, UK, pp. 220–232.
Stepanek, L., Stoker, A. W., Stoeckli, E., and Bixby, J. L. (2005). Receptor tyrosine phosphatases guide vertebrate motor axons during development. J. Neurosci. 25, 3813–3823.
Dai, F., Yusuf, F., Farjah, G. H., and Brand-Saberi, B. (2005). RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis. Dev. Biol. 285, 80–90.
Sato, F., Nakagawa, T., Ito, M., Kitagawa, Y., and Hattori, M. A. (2004). Application of RNA interference to chicken embryos using small interfering RNA. J. Exp. Zoolog. A Comp. Exp. Biol. 301, 820–827.
Perrin, F. E., and Stoeckli, E. T. (2000). Use of lipophilic dyes in studies of axonal pathfinding in vivo. Microsc. Res. Tech. 48, 25–31.
Matsuda, T., and Cepko, C. L. (2004). Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. USA 101, 16–22.
Oberg, K. C., Pira, C. U., Revelli, J. P., Ratz, B., Aguilar-Cordova, E., and Eichele, G. (2002). Efficient ectopic gene expression targeting chick mesoderm. Dev. Dyn. 224, 291–302.
Luo, J., and Redies, C. (2004). Overexpression of genes in Purkinje cells in the embryonic chicken cerebellum by in vivo electroporation. J. Neurosci. Meth. 139, 241–245.
Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., and Funahashi, J. (2004). Gain- and loss-of-function in chick embryos by electroporation. Mech. Dev. 121, 1137–1143.
Billy, E., Brondani, V., Zhang, H., Muller, U., and Filipowicz, W. (2001). Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc. Natl. Acad. Sci. USA 98, 14428–14433.
Stein, P., Zeng, F., Pan, H., and Schultz, R. M. (2005). Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes. Dev. Biol. 286, 464–471.
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Baeriswyl, T., Mauti, O., Stoeckli, E.T. (2008). Temporal Control of Gene Silencing by in ovo Electroporation. In: Barik, S. (eds) RNAi. Methods in Molecular Biology™, vol 442. Humana Press. https://doi.org/10.1007/978-1-59745-191-8_16
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DOI: https://doi.org/10.1007/978-1-59745-191-8_16
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