Efficient Gene Delivery into Multiple CNS Territories Using <em>In Utero</em> Electroporation

Abstract

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 embryos(1,2) that we have successfully adapted(3,4). It is largely based on the success of in ovo electroporation technologies that are commonly used in chick(5). 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 described(6). 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 development(1,2,3,4). More recent studies have targeted the embryonic retina(7,8,9) and thalamus(10,11,12). 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 3(rd) 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.

Neurog1andNeurog2Control Two Waves of Neuronal Differentiation in the Piriform Cortex

“…1K-M'). This is consistent with the pallial identity of CR neurons (Hevner et al, 2003) and with our previous shortterm lineage-tracing experiments in E11.5 Neurog2 GFP KI/ϩ neocortices (Dixit et al, 2011b).…”

“…Neurog2-derived CR cells are widespread, populating the piriform cortex, neocortex, and hippocampus (this study; Dixit et al, 2011b). It is thus not surprising that we observed a global reduction in CR cell numbers in all cortical domains in Neurog1/2 Ϫ/Ϫ embryos.…”

“…Neurog1 and Neurog2 are expressed in CR cell lineages derived from all three progenitor domains (this study; Dixit et al, 2011b), including the rostrobulbar area/pallial septum, cortical hem/choroid plexus, and ventral pallium (Takiguchi-Hayashi et al, 2004;Bielle et al, 2005;Yoshida et al, 2006;Zhao et al, 2006;García-Moreno et al, 2007;Imayoshi et al, 2008). Neurog2-derived CR cells are widespread, populating the piriform cortex, neocortex, and hippocampus (this study; Dixit et al, 2011b).…”

Induction of Protein Deletion Through <em>In Utero</em> Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

“…In utero electroporation (IUE) is the process by which a plasmid carrying both a gene of interest and a reporter is injected into the ventricle in the brain of a mouse or rat embryo and then drawn into the cells lining the ventricle through use of an electric current [12][13][14] . This allows the investigator to analyze the effect of up or down regulation of a gene of interest in development or function of electroporated neurons.…”

Expansion of Embryonic and Adult Neural Stem Cells by <em>In Utero</em> Electroporation or Viral Stereotaxic Injection

“…By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9 . Both approaches, whose basic principles were already described by other video protocols [10][11][12][13][14] , were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e.…”