Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Written By India Gate on Tuesday, June 28, 2011 | 1:02 AM


The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.


In utero electroporation can be used to analyze a wide variety of developmental processes. For example, transfection of reporter genes such as GFP, mCherry or alkaline phosphatase can be used to conduct lineage tracing and neuronal migration experiments. Alternatively, Cre recombinase can be transiently expressed to selectively eliminate a floxed allele in a spatially- and/or temporally-controlled manner. Furthermore, shRNA or dominant negative constructs can be electroporated to knockdown target gene function. Finally, targeted overexpression or misexpression of key genes in both wild type and/or genetically mutant mouse lines can be used to study cell fate decisions. The high throughput of this assay is critical as it allows for the testing of many combinations of factors in a very short time. One note of caution is that this procedure does cause changes in gene expression in cells that line the needle entry site (i.e., injury-response genes upregulated in wound; as observed by us and others13). It is thus recommended to focus on electroporated cells outside of the wound site. In addition, embryonic survival rates are low when first learning this technique but quickly rise to >95% with practice. To date, we have successfully used in utero electroporation technologies to identify genes regulated by proneural bHLH transcription factors in the telencephalon4. We have also validated the use of this technique for the analysis of telencephalic cis-regulatory elements3.


No conflicts of interest declared.


The authors would like to thank Eva Hadzimova, Pierre Mattar and Christopher Kovach for their initial work in establishing in utero electroporation technology in the CS lab. This work was funded by a Canadian Institute of Health Research (CIHR) grant (MOP 44094) and CIHR/Foundation Fighting Blindness (FFB) Emerging Team Grant (00933-000) to CS and an Alberta Children’s Hospital Research Foundation Grant to DMK. RD was supported by a CIHR Canada Hope Scholarship, RC is supported by an FFB Studentship and LML was supported by a CIHR Training Grant in Genetics and Child Development.


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  1. Saito, T., & Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240, 237-246, doi:10.1006/dbio.2001.0439 S0012-1606(01)90439-7 [pii] (2001).
  2. Takahashi, M., Sato, K., Nomura, T., & Osumi, N. Manipulating gene expressions by electroporation in the developing brain of mammalian embryos. Differentiation 70, 155-162, doi:S0301-4681(09)60445-X [pii] 10.1046/j.1432-0436.2002.700405.x (2002).
  3. Langevin, L. M. et al. Validating in utero electroporation for the rapid analysis of gene regulatory elements in the murine telencephalon. Dev Dyn 236, 1273-1286, doi:10.1002/dvdy.21126 (2007).
  4. Mattar, P. et al. Basic helix-loop-helix transcription factors cooperate to specify a cortical projection neuron identity. Mol Cell Biol 28, 1456-1469, doi:MCB.01510-07 [pii] 10.1128/MCB.01510-07 (2008).
  5. Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., & Funahashi, J. Gain- and loss-of-function in chick embryos by electroporation. Mech Dev 121, 1137-1143, doi:10.1016/j.mod.2004.05.013 S092547730400139X [pii] (2004).
  6. Gaiano, N., Kohtz, J. D., Turnbull, D. H., & Fishell, G. A method for rapid gain-of-function studies in the mouse embryonic nervous system. Nat Neurosci 2, 812-819, doi:10.1038/12186 (1999).
  7. Matsuda, T., & Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A 101, 16-22, doi:10.1073/pnas.2235688100 2235688100 [pii] (2004).
  8. Petros, T. J., Rebsam, A., & Mason, C. A. In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells. J Vis Exp, doi:1333 [pii] 10.3791/1333 (2009).
  9. Garcia-Frigola, C., Carreres, M. I., Vegar, C., & Herrera, E. Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev Biol 7, 103, doi:1471-213X-7-103 [pii] 10.1186/1471-213X-7-103 (2007).
  10. Kataoka, A., & Shimogori, T. Fgf8 controls regional identity in the developing thalamus. Development 135, 2873-2881, doi:dev.021618 [pii] 10.1242/dev.021618 (2008).
  11. Vue, T. Y. et al. Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice. J Neurosci 29, 4484-4497, doi:29/14/4484 [pii] 10.1523/JNEUROSCI.0656-09.2009 (2009).
  12. Tsuchiya, R., Takahashi, K., Liu, F.C., & Takahashi, H. Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections. J Neurosci Res 87, 1620-1633, doi:10.1002/jnr.21966 (2009).
  13. Buffo, A. et al. Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair. Proc Natl Acad Sci U S A 102, 18183-18188, doi:0506535102 [pii] 10.1073/pnas.0506535102 (2005).

Cite this Article

Dixit, R., Lu, F., Cantrup, R., Gruenig, N., Langevin, L. M., Kurrasch, D. M., Schuurmans, C., Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation. http://www.jove.com/details.php?id=2957 doi: 10.3791/2957. J Vis Exp. 52 (2011).