Abstract
This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.
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Change history
29 December 2006
In the version of this article initially published online, several instances of the phrase in vivo were incorrectly substituted with in ovo. When the article was first corrected, not all instances were changed appropriately. These errors have been corrected in all versions of the article. In addition, on p. 1557 the first line of the Troubleshooting section should read “Steps 6A(i)–(iv) and 6B(i)–(iv)…” rather than “Steps 6A(i)–(iv) and 6B(ix)–(xii)…”
References
Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo: A Laboratory Manual 3rd edn. (Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY, USA, 2003).
Gaensler, K.M.L. et al. Fetal gene transfer by transuterine injection of cationic liposome–DNA complexes. Nature Biotechnol. 17, 1188–1192 (1999).
Yang, N.-S. & Sun, W.H. Gene gun and other non-viral approaches for cancer gene therapy. Nature Med. 1, 481–483 (1995).
Calvin, N.M. & Hanawalt, P.C. High-efficiency transformation of bacterial cells by electroporation. J. Bacteriol. 170, 2796–2801 (1988).
Neumann, E., Schaefer-Ridder, M., Wang, Y. & Hofschneider, P.H. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1, 841–845 (1982).
Fromm, M., Taylor, L.P. & Walbot, V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82, 5824–5828 (1985).
Aihara, H. & Miyazaki, J. Gene transfer into muscle by electroporation in vivo . Nature Biotechnol. 16, 867–870 (1998).
Muramatsu, T., Mizutani, Y., Ohmori, Y. & Okumura, J. Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo . Biochem. Biophys. Res. Comm. 230, 376–380 (1997).
Itasaki, N., Bel-Vialar, S. & Krumlauf, R. 'Shocking' developments in chick embryology: electroporation and in ovo gene expression. Nature Cell Biol. 1, E203–E207 (1999).
Saito, T. & Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237–246 (2001).
Saba, R., Nakatsuji, N. & Saito, T. Mammalian BarH1 confers commissural neuron identity on dorsal cells in the spinal cord. J. Neurosci. 23, 1987–1991 (2003).
Ding et al. Lmx1b controls the differentiation and migration of the superficial dorsal horn neurons of the spinal cord. Development 131, 3693–3703 (2004).
Hasegawa, H. et al. Laminar patterning in the developing neocortex by temporally coordinated fibroblast growth factor signaling. J. Neurosci. 24, 8711–8719 (2004).
Saba, R., Johnson, J.E. & Saito, T. Commissural neuron identity is specified by a homeodomain protein, Mbh1, that is directly downstream of Math1. Development 132, 2147–2155 (2005).
Molyneaux, B.J. et al. Fezl is required for the birth and specification of corticospinal motor neurons. Neuron 47, 817–831 (2005).
Nguyen, L. et al. p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex. Genes Dev. 20, 1511–1524 (2006).
Miyagi, S. et al. The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells. Mol. Cell. Biol. 24, 4207–4220 (2004).
Mizutani, K. & Saito, T. Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex. Development 132, 1295–1304 (2005).
Borell, V., Yoshimura, Y. & Callaway, E.M. Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J. Neurosci. Methods 143, 151–158 (2005).
Kawauchi, D. et al. Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration. Development 133, 1113–1123 (2006).
Pekarik, V. et al. Screening for gene function in chicken embryo using RNAi and electroporation. Nature Biotechnol. 21, 93–96 (2003).
Bai, J. et al. RNAi reveals doublecortin is required for radial migration in rat neocortex. Nature Neurosci. 6, 1277–1283 (2003).
Takeuchi, A. & O'Leary, D.M. Radial migration of superficial layer cortical neurons controlled by novel Ig cell adhesion molecule MDGA1. J. Neurosci. 26, 4460–4464 (2006).
Huang, Z. et al. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene. FEBS Lett. 487, 248–251 (2000).
Matsuda, T. & Cepko, C.L. Electroporation and RNA interference in the rodent retina in vivo and in vitro . Proc. Natl. Acad. Sci. USA 101, 16–22 (2004).
Muneoka, K., Wanek, N., Trevino, C. & Bryant, S.V. Exo utero surgery. in Postimplantation Mammalian Embryos: A Practical Approach (eds. Copp, A.J. & Cockroft, D.L.) 41–59 (Oxford University Press, Oxford, UK, 1990).
Acknowledgements
I thank J. Kutsuna, M. Tanaka and Y. Yamamoto for their help and Dr. D. Kawauchi for critical reading of the manuscript. This work was supported by Brain Science Foundation and grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Saito, T. In vivo electroporation in the embryonic mouse central nervous system. Nat Protoc 1, 1552–1558 (2006). https://doi.org/10.1038/nprot.2006.276
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DOI: https://doi.org/10.1038/nprot.2006.276
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