We thank Dr. G. McGillivray, Pr. J. Clayton-Smith, Pr. W.B. Dobyns, Pr. P. Striano, Pr. I.E. Scheffer, Pr. S.P. Roberston and Pr. S.F. Berkovic for providing MCD patients. We thank Dr. F. Michel and D. Mei for technical advices and help. This work was supported by funding from the Seven Framework Programme of the EU, DESIRE project, contract number: Health-F2-602531-2013, (to V.C., R.G., A.R., A.F. and C.C.), INSERM (to A.R. and C.C.), Fondation J&#233;r&#244;me Lejeune (R13083AA to A.F, E.P.P and C.C) and R&#233;gion Provence Alpes C&#244;te d&#39;Azur (APO2014 - DEMOTIC to C.C. and A.A.D). D.A.K is an EMBO Young Investigator and is supported by FWF grants (I914 and P24367).

<ol><li>Meencke, H. J., Veith, G. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/1285910">Migration disturbances in epilepsy.</a> Epilepsy Res. 9, 31-39 (1992).</li>
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<li>Dobyns, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Consistent+chromosome+abnormalities+identify+novel+polymicrogyria+loci+in+1p36.3%2C+2p16.1-p23.1%2C+4q21.21-q22.1%2C+6q26-q27%2C+and+21q2%5BTitle%5D)+AND+%22Am+J+Med+Genet+A%22%5BJournal%5D)">Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2.</a> Am J Med Genet A. 146 (13), 1637-1654 (2008).</li>
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</ol>

The authors have nothing to disclose.

MCDs are important causes of intellectual disability and account for 20-40% of drug-resistant childhood epilepsy1,2. Interest in MCDs has increased dramatically over the past decade as a result of two major factors. The first is the improvement in brain imaging (particularly MRI), which allows physicians and scientists to visualize many brain malformations that were not previously recognized. The other is the evolution of genetic tools that have allowed the ident...

The cerebral cortex plays a key role in cognitive and intellectual processes and is involved in emotional control as well as learning and memory. It is therefore not surprising that many neurological and psychiatric diseases result from malformations of cortical development (MCD). The etiology of MCD is complex since both acquired and genetic factors are involved. The cumulative prevalence of genetically determined proportion of MCD is about 2% and they are sporadic in most cases. For instance, the incidence of congenital brain dysgenesis was estimated to be higher than 1% in the human population, and some forms of MCD are observed in more than 14% of all patients with epilepsy and in 40% of severe or intractable epilepsy1,2.
Periventricular Nodular Heterotopia (PNH) is one of the most common MCDs and is caused by abnormal migration of neurons from the ventricular zone (VZ) to the developing cerebral cortex. The failure of neurons to migrate results in clusters of heterotopic neurons along the walls of the lateral ventricles which can usually be visualized using Magnetic Resonance Imaging (MRI). The clinical, anatomic and imaging features of PNH are heterogeneous. Nodules may range from small and unilateral to bilateral and symmetric. Common clinical sequelae include epilepsy and intellectual disabilities3. Mutations in the Filamin A (or FLNA) gene, which maps in Xq28, were found in 100% of families with X-linked bilateral PNH and in 26% of sporadic patients3,4. A rare, recessive form of PNH caused by mutations in the ARFGEF2 gene, which maps in 20q13, has been reported in two consanguineous families5. Recently, biallelic mutations in genes encoding the receptor-ligand cadherin pair DCHS1 and FAT4 have been identified in nine patients affected by a multisystemic disorder that includes PNH6. PNH has also been associated with fragile X syndrome7, Williams syndrome8, 22q11 microdeletion syndrome9, duplications at 5p1510, deletions at 1p3611, 5q14.3-q1512, 6p2513 and 6q terminal deletion syndrome14,15,16,17,18,19, suggesting that additional causative genes are scattered throughout the genome. However, for approximately 74% of sporadic PNH patients the genetic basis remains to be elucidated17.
Classical gene mapping approaches such as array Comparative Genomic Hybridization (array-CGH) have proven to be a powerful tools for the detection of sub-microscopic chromosomal abnormalities, however, the genomic regions identified using this approach are often large and contain numerous genes.
The advent of massive parallel sequencing techniques (i.e. Whole-Exome Sequencing (WES) and Whole-Genome sequencing (WGS)) has substantially reduced both the cost and the time required to sequence an entire human exome or genome. Nevertheless, interpretation of WES and WGS data remains challenging in the majority of cases, since for each patient tens to hundreds (or even thousands, depending from the type of analysis) variants emerge from data filtering.
To speed up the process of identifying novel MCD causative genes, a novel systematic strategy combining array-CGH, WES and in utero electroporation (IUE) screening of candidate genes was designed. IUE allows to selectively inactivate (or overexpress) specific genes or mutations in rodent brains, enabling rapid evaluation of their involvement in corticogenesis18,19. RNAi mediated-knockdown or overexpression of one or more candidate genes is expected to cause, when the gene is associated with disease development, localized defects in neuronal migration and/or maturation. Upon the identification of a gene whose inactivation (or overexpression) reproduces the phenotype observed in patients in rodents, it becomes an outstanding candidate for the screening in sporadic patients with MCD. Using this approach, we recently revealed the crucial contribution of the C6orf70 gene (also known as ERMARD) in PNH pathogenesis in patients harbouring 6q27 chromosomal deletions16.

<table><tbody><tr><td>Picospritzer III</td><td>Parker Hannifin Corp</td><td>P/N 051-0500-900</td><td>Intracellular Microinjection Dispense Systems</td></tr><tr><td>Fast Green FCF</td><td>Sigma-Aldrich</td><td>F7252</td><td>Fast green allow visual monitoring of the injection</td></tr><tr><td>BTX ECM 830 electroporator</td><td>BTX Harvard Apparatus</td><td>45-0002</td><td>The ECM 830 is a Square Wave Pulse generator designed for in vitro and in vivo applications</td></tr><tr><td>Microtome HM 650 V</td><td>Microm</td><td>10076838</td><td>Microtome HM 650 V vibratome 240V 50/60Hz with vibrating blade</td></tr><tr><td>FluoView 300</td><td>Olympus</td><td></td><td>The Olympus FluoViewTM 300 is a point-scanning, point-detection, confocal laser scanning microscope designed for biology research application</td></tr><tr><td>eCELLence software</td><td>Glance Vision Technologies</td><td></td><td>eCELLence is software designed for the quantitive analysis of cell migration</td></tr><tr><td>Agilent Microarray Scanner Bundle</td><td></td><td></td><td>Array slides</td></tr><tr><td>for 1 x 244K, 2 x 105K, 4 x 44K or 8 x 15K</td><td>Agilent</td><td>Agilent p/n G4900DA, G2565CA or G2565BA</td><td></td></tr><tr><td>for 1 x 1M, 2 x 400K, 4 x 180K or 8 x 60K</td><td>Agilent</td><td>Agilent p/n G4900DA or G2565CA</td><td></td></tr><tr><td>Hybridization Chamber, stainless</td><td>Agilent</td><td>Agilent p/n G2534A</td><td>Chamber for array CGH hybridization</td></tr><tr><td>Hybridization Chamber gasket slides, 5-pack</td><td></td><td></td><td>Gasket for array CGH hybridization</td></tr><tr><td>for 1-pack microarrays or</td><td>Agilent</td><td>Agilent p/n G2534-60003</td><td></td></tr><tr><td>for 2-pack microarrays or</td><td>Agilent</td><td>Agilent p/n G2534-60002</td><td></td></tr><tr><td>for 4-pack microarrays or</td><td>Agilent</td><td>Agilent p/n G2534-60011</td><td></td></tr><tr><td>for 8-pack microarrays</td><td>Agilent</td><td>Agilent p/n G2534-60014</td><td></td></tr><tr><td>Hybridization oven</td><td>Agilent</td><td>Agilent p/n G2545A</td><td></td></tr><tr><td>Hybridization oven rotator for Agilent Microarray Hybridization Chambers</td><td>Agilent</td><td>Agilent p/n G2530-60029</td><td></td></tr><tr><td>Thermal cycler with heated lid</td><td>Agilent</td><td>Agilent p/n G8800A or equivalent</td><td>Termal cycler for incubations</td></tr><tr><td>1.5 mL RNase-free Microfuge Tube</td><td>Ambion</td><td>p/n AM12400 or equivalent</td><td>Microcentrifuge</td></tr><tr><td>Magnetic stir bar</td><td>Corning</td><td>p/n 401435 or equivalent</td><td>Instrument for stirring</td></tr><tr><td>Qubit Fluorometer</td><td>Life Technologies</td><td>p/n Q32857</td><td>Instrument for DNA quantification</td></tr><tr><td>Qubit dsDNA BR Assay Kit, for use with the Qubit fluorometer</td><td>Invitrogen</td><td>p/n Q32850</td><td>Kit for Qubit fluorometer</td></tr><tr><td>UV-VIS spectrophotometer</td><td>Thermo Scientific</td><td>NanoDrop 8000 or 2000, or equivalent</td><td>Instrument for DNA quantification</td></tr><tr><td>P10, P20, P200 and P1000 pipettes</td><td>Pipetman or equivalent</td><td></td><td>DNA dispensation</td></tr><tr><td>Vacuum Concentrator</td><td>Thermo Scientific</td><td>p/n DNA120-115 or&lt;br/&gt; equivalent</td><td>Instrument to concentrate DNA</td></tr><tr><td>SureTag Complete DNA Labeling Kit</td><td>Agilent</td><td>p/n 5190-4240</td><td>DNA Labeling Kit (for Human Samples)</td></tr><tr><td>Purification Columns (50 units)</td><td>Agilent</td><td>p/n 5190-3391</td><td>DNA Labeling Kit (for Human Samples)</td></tr><tr><td>AutoScreen A, 96-well plates</td><td>GE Healthcare</td><td>p/n 25-9005-98</td><td>DNA Labeling Kit (for Human Samples)</td></tr><tr><td>GenElute PCR Clean-Up Kit</td><td>Sigma-Aldrich</td><td>p/n NA1020</td><td>DNA Labeling Kit (for Human Samples)</td></tr><tr><td>Human Genomic DNA</td><td></td><td>p/n G1521</td><td>DNA Labeling Kit (for Human Samples)</td></tr><tr><td>For CGH microarrays:</td><td>Promega</td><td>(female) or p/n G1471 (male)</td><td>Array CGH control DNA</td></tr><tr><td>For CGH+SNP microarrays:</td><td>Coriell</td><td>p/n NA18507, NA18517, NA12891, NA12878, or NA18579</td><td>Array CGH control DNA</td></tr><tr><td>Oligo aCGH/ChIP-on-chip Wash Buffer Kit or</td><td>Agilent</td><td>p/n 5188-5226</td><td>Array CGH hybridization and wash</td></tr><tr><td>Oligo aCGH/ChIP-on-chip Wash Buffer 1 and</td><td>Agilent</td><td>p/n 5188-5221</td><td>Array CGH hybridization and wash</td></tr><tr><td>Oligo aCGH/ChIP-on-chip Wash Buffer 2</td><td>Agilent</td><td>p/n 5188-5222</td><td>Array CGH hybridization and wash</td></tr><tr><td>Stabilization and Drying Solution</td><td>Agilent</td><td>p/n 5185-5979</td><td>Array CGH hybridization and wash</td></tr><tr><td>Oligo aCGH/ChIP-on-chip Hybridization Kit</td><td>Agilent</td><td>p/n 5188-5220 (25) or p/n 5188-5380 (100)</td><td>Array CGH hybridization and wash</td></tr><tr><td>Human Cot-1 DNA</td><td>Agilent</td><td>p/n 5190-3393</td><td>Array CGH hybridization and wash</td></tr><tr><td>Agilent C scanner</td><td>Agilent</td><td></td><td>Scanner for array CGH slides</td></tr><tr><td>SureSelect XT2 Reagent Kit</td><td></td><td></td><td>Kit for target enrichment</td></tr><tr><td>HiSeq platform (HSQ), 16 Samples</td><td>Agilent</td><td>p/n G9621A</td><td></td></tr><tr><td>HiSeq platform (HSQ), 96 Samples</td><td>Agilent</td><td>p/n G9621B</td><td></td></tr><tr><td>HiSeq platform (HSQ), 480 Samples</td><td>Agilent</td><td>p/n G9621C</td><td></td></tr><tr><td>MiSeq platform (MSQ), 16 Samples</td><td>Agilent</td><td>p/n G9622A</td><td></td></tr><tr><td>MiSeq platform (MSQ), 96 Samples</td><td>Agilent</td><td>p/n G9622B</td><td></td></tr><tr><td>MiSeq platform (MSQ), 480 Samples</td><td>Agilent</td><td>p/n G9622C</td><td></td></tr><tr><td>DNA 1000 Kit</td><td>Agilent</td><td>p/n 5067-1504</td><td>Kit for the separation, sizing and quantification of dsDNA fragments from 25 to 1000 bp.</td></tr><tr><td>High Sensitivity DNA Kit</td><td>Agilent</td><td>p/n 5067-4626</td><td>Kit for analysis of fragmented DNA or DNA libraries.</td></tr><tr><td>AMPure XP Kit</td><td></td><td></td><td>Kit for automated PCR purification.</td></tr><tr><td>5 mL</td><td>Agencourt</td><td>p/n A63880</td><td></td></tr><tr><td>60 mL</td><td>Agencourt</td><td>p/n A63881</td><td></td></tr><tr><td>450 mL</td><td>Agencourt</td><td>p/n A63882</td><td></td></tr><tr><td>Dynabeads MyOne Streptavidin T1</td><td></td><td></td><td>Isolation and handling of biotinylated nucleic acids</td></tr><tr><td>2 mL</td><td>Life Technologies</td><td>Cat #65601</td><td></td></tr><tr><td>10 mL</td><td>Life Technologies</td><td>Cat #65602</td><td></td></tr><tr><td>Quant-iT dsDNA BR Assay Kit, for the Qubit fluorometer</td><td></td><td></td><td>DNA quantification</td></tr><tr><td>100 assays, 2-1000 ng</td><td>Life Technologies</td><td>Cat #Q32850</td><td></td></tr><tr><td>500 assays, 2-1000 ng</td><td>Life Technologies</td><td>Cat #Q32853</td><td></td></tr><tr><td>Qubit assay tubes</td><td>Life Technologies</td><td>p/n Q32856</td><td>DNA quantification</td></tr><tr><td>SureSelec tXT2 Capture Libraries</td><td>Agilent</td><td>depending on the experiment</td><td>Kit for libraries capture</td></tr><tr><td>SureCycler 8800 Thermal Cycler</td><td>Agilent</td><td>p/n G8800A</td><td>DNA amplification</td></tr><tr><td>96 well plate module for SureCycler 8800 Thermal Cycler</td><td>Agilent</td><td>p/n G8810A</td><td>DNA amplification</td></tr><tr><td>SureCycler 8800-compatible 96-well plates</td><td>Agilent</td><td>p/n 410088</td><td>DNA amplification</td></tr><tr><td>Optical strip caps</td><td>Agilent</td><td>p/n 401425</td><td>DNA amplification</td></tr><tr><td>Tube cap strips, domed</td><td>Agilent</td><td>p/n 410096</td><td>DNA amplification</td></tr><tr><td>Compression mats</td><td>Agilent</td><td>p/n 410187</td><td>DNA amplification</td></tr><tr><td>2100 Bioanalyzer Laptop Bundle</td><td>Agilent</td><td>p/n G2943CA</td><td>DNA amplification</td></tr><tr><td>2100 Bioanalyzer Electrophoresis Set</td><td>Agilent</td><td>p/n G2947CA</td><td>DNA amplification</td></tr><tr><td>Covaris Sample Preparation System, E-series or S-series</td><td>Covaris</td><td></td><td>DNA shearing</td></tr><tr><td>Covaris sample holders</td><td></td><td>p/n 520078</td><td>DNA shearing</td></tr><tr><td>Nutator plate mixer</td><td>BD Diagnostics</td><td>p/n 421105 or equivalent</td><td>Plate Mixer</td></tr><tr><td>GaIIx</td><td>Illumina</td><td></td><td>next generation sequencing machine</td></tr></tbody></table>

a novel strategy combining array-cgh, whole-exome sequencing and in utero electroporation in rodents to identify causative genes for brain malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

The experimental strategy designed to identify novel MCD causative genes is recapitulated in Figure 1.
By performing array-CGH in a cohort of 155 patients with developmental brain abnormalities variably combining PNH (Figure 2A), corpus callosum dysgenesis, colpocephaly, cerebellar hypoplasia and polymicrogyria associated with epilepsy, ataxia and cognitive impairment, we identified a 1.2 Mb min...

Watch this Scientific Journal Video about A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations at Jo

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

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Research

JoVE Journal

Neuroscience

8.6K Views.  University of Florence. Nodulair heterotopia van de Periventricular (PNH) is de meest voorkomende vorm van misvorming van corticale ontwikkeling (MCD) in volwassenheid maar haar genetische basis blijft onbekend in meest sporadische gevallen. Onlangs hebben we een strategie nieuwe kandidaat om genen te identificeren voor MCDs en direct bevestigen hun oorzakelijke rol in vivo.

Video: A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

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

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

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

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

In Vivo Brain Tumor Modeling via Electroporation-Based Delivery of Plasmid DNA

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

Author Spotlight: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures  

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...

Cancer Research

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

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