Nous remercions Lisa Kretsge pour les commentaires critiques et l’édition du manuscrit. Nous remercions tous les assistants de recherche du laboratoire Cruz-Martín qui ont été précieux pour aider aux perfusions et au comptage cellulaire des cerveaux comportementaux. Nous remercions Andrzej Cwetsch pour son apport sur la conception de l’électrode tripolar, et Todd Blute et le centre d’imagerie en biologie de l’Université de Boston pour l’utilisation du microscope confocal. Ces travaux ont été appuyés par une subvention pour les jeunes chercheurs de la NARSAD (AC-M, #27202), le Prix Brenton R. Lutz (ALC), le Prix I. Alden Macchi (ALC), le NSF NRT UtB: Neurophotonics National Research Fellowship (ALC, #DGE1633516) et le Boston University Undergraduate Research Opportunities Program (WWY). Les bailleurs de fonds n’ont joué aucun rôle dans la conception, la collecte et l’analyse des données, la décision de publier ou la préparation du manuscrit.

<ol>
	<li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+association+and+meta-analysis+of+a+schizophrenia+GWAS+variant+rs10489202+in+East+Asian+populations.">Genetic association and meta-analysis of a schizophrenia GWAS variant rs10489202 in East Asian populations.</a> Translational Psychiatry. 8 (1), 144 (2018).</li><li>Schizophrenia Working Group of the Psychiatric Genomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+insights+from+108+schizophrenia-associated+genetic+loci.">Biological insights from 108 schizophrenia-associated genetic loci.</a> Nature. 511 (7510), 421-427 (2014).</li><li>Howard, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+meta-analysis+of+depression+identifies+102+independent+variants+and+highlights+the+importance+of+the+prefrontal+brain+regions.">Genome-wide meta-analysis of depression identifies 102 independent variants and highlights the importance of the prefrontal brain regions.</a> Nature Neuroscience. 22 (3), 343-352 (2019).</li><li>Grove, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+common+genetic+risk+variants+for+autism+spectrum+disorder.">Identification of common genetic risk variants for autism spectrum disorder.</a> Nature Genetics. 51 (3), 431-444 (2019).</li><li>Comer, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+schizophrenia-associated+gene+C4+leads+to+hypoconnectivity+of+prefrontal+cortex+and+reduced+social+interaction.">Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction.</a> PLoS Biology. 18 (1), 3000604 (2020).</li><li>Chini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+and+Rescuing+Developmental+Miswiring+in+a+Mouse+Model+of+Cognitive+Impairment.">Resolving and Rescuing Developmental Miswiring in a Mouse Model of Cognitive Impairment.</a> Neuron. 105 (1), 60-74 (2020).</li><li>Clifton, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+expression+of+genes+associated+with+schizophrenia+and+bipolar+disorder+across+development.">Dynamic expression of genes associated with schizophrenia and bipolar disorder across development.</a> Translational Psychiatry. 9 (1), 74 (2019).</li><li>Oberlander, V. C., Xu, X., Chini, M., Hanganu-Opatz, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+dysfunction+of+prefrontal-hippocampal+networks+in+mouse+models+of+mental+illness.">Developmental dysfunction of prefrontal-hippocampal networks in mouse models of mental illness.</a> European Journal of Neurosciences. 50 (6), 3072-3084 (2019).</li><li>Batista-Brito, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+Dysfunction+of+VIP+Interneurons+Impairs+Cortical+Circuits.">Developmental Dysfunction of VIP Interneurons Impairs Cortical Circuits.</a> Neuron. 95 (4), 884-895 (2017).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Dissection+of+Neural+Circuits:+A+Decade+of+Progress.">Genetic Dissection of Neural Circuits: A Decade of Progress.</a> Neuron. 98 (2), 256-281 (2018).</li><li>DeNardo, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+strategies+to+access+activated+neurons.">Genetic strategies to access activated neurons.</a> Current Opinion in Neurobiology. 45, 121-129 (2017).</li><li>Huang, Z. J., Zeng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+approaches+to+neural+circuits+in+the+mouse.">Genetic approaches to neural circuits in the mouse.</a> Annual Reviews in Neurosciences. 36, 183-215 (2013).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Molecular Therapy. 18 (1), 80-86 (2010).</li><li>Callaway, E. M., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+Circuit+Tracing+with+Glycoprotein-Deleted+Rabies+Viruses.">Monosynaptic Circuit Tracing with Glycoprotein-Deleted Rabies Viruses.</a> Journal of Neurosciences. 35 (24), 8979-8985 (2015).</li><li>Cruz-Mart&#237;n, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dedicated+circuit+links+direction-selective+retinal+ganglion+cells+to+the+primary+visual+cortex.">A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex.</a> Nature. 507 (7492), 358-361 (2014).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nature Protocols. 1 (3), 1552-1558 (2006).</li><li>Cruz-Mart&#237;n, A., Crespo, M., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+stabilization+of+dendritic+spines+in+fragile+X+mice.">Delayed stabilization of dendritic spines in fragile X mice.</a> Journal of Neurosciences. 30 (23), 7793-7803 (2010).</li><li>Zhang, J. H., Adikaram, P., Pandey, M., Genis, A., Simonds, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+genome+editing+through+CRISPR-Cas9+engineering.">Optimization of genome editing through CRISPR-Cas9 engineering.</a> Bioengineered. 7 (3), 166-174 (2016).</li><li>Strecker, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+DNA+insertion+with+CRISPR-associated+transposases.">RNA-guided DNA insertion with CRISPR-associated transposases.</a> Science. 365 (6448), 48-53 (2019).</li><li>Cruz-Mart&#237;n, A., Crespo, M., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+induces+the+elongation+of+early+dendritic+protrusions+via+mGluRs+in+wild+type+mice,+but+not+in+fragile+X+mice.">Glutamate induces the elongation of early dendritic protrusions via mGluRs in wild type mice, but not in fragile X mice.</a> PLoS One. 7 (2), 32446 (2012).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cerebral Cortex. 19, 120-125 (2009).</li><li>Kolk, S. M., de Mooij-Malsen, A. J., Martens, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+Molecular+Approach+of+in+utero+Electroporation+to+Functionally+Decipher+Endophenotypes+in+Neurodevelopmental+Disorders.">Spatiotemporal Molecular Approach of in utero Electroporation to Functionally Decipher Endophenotypes in Neurodevelopmental Disorders.</a> Frontiers in Molecular Neurosciences. 4, 37 (2011).</li><li>Szczurkowska, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+in+vivo+genetic+manipulation+of+the+mouse+or+rat+brain+by+in+utero+electroporation+with+a+triple-electrode+probe.">Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe.</a> Nature Protocols. 11 (3), 399-412 (2016).</li><li>dal Maschio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+and+site-directed+in+utero+electroporation+by+a+triple-electrode+probe.">High-performance and site-directed in utero electroporation by a triple-electrode probe.</a> Nature Communications. 3, 960 (2012).</li><li>Zhan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficient+neuron-microglia+signaling+results+in+impaired+functional+brain+connectivity+and+social+behavior.">Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior.</a> Nature Neurosciences. 17 (3), 400-406 (2014).</li><li>Mathis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeepLabCut:+markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning.">DeepLabCut: markerless pose estimation of user-defined body parts with deep learning.</a> Nature Neurosciences. 21 (9), 1281-1289 (2018).</li><li>Bullmann, T., Arendt, T., Frey, U., Hanashima, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transportable,+inexpensive+electroporator+for+in+utero+electroporation.">A transportable, inexpensive electroporator for in utero electroporation.</a> Development Growth and Differentiation. 57 (5), 369-377 (2015).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+analysis+identifies+30+new+susceptibility+loci+for+schizophrenia.">Genome-wide association analysis identifies 30 new susceptibility loci for schizophrenia.</a> Nature Genetics. 49 (11), 1576-1583 (2017).</li><li>Ripke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+analysis+identifies+13+new+risk+loci+for+schizophrenia.">Genome-wide association analysis identifies 13 new risk loci for schizophrenia.</a> Nature Genetics. 45 (10), 1150-1159 (2013).</li><li>De Marco Garcia, N. V., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-selective+electroporation+of+cortical+interneurons.">Subtype-selective electroporation of cortical interneurons.</a> Journal of Visualized Experiment. (90), e51518 (2014).</li><li>Borrell, V., Yoshimura, Y., Callaway, E. 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Les auteurs ne déclarent aucun intérêt concurrent.

Dans ce cas, un pipeline est décrit qui combine la manipulation de nouveaux gènes d’intérêt dans de grandes populations de neurones corticals frontaux avec des analyses comportementales chez la souris. En outre, ce pipeline permet l’étude longitudinale du comportement chez les mêmes souris à la fois au cours du développement postnatal précoce et à l’âge adulte. Cette technique contourne la nécessité de s’appuyer sur des modèles animaux génétiques qui peuvent être coûteux en termes de temps et de...

Les études d’association à l’échelle du génome (GWAS) ont conduit à la découverte de nouveaux gènes candidats associés aux pathologies cérébrales1,2,3,4. Ces études ont été particulièrement bénéfiques pour comprendre les troubles neuropsychiatriques dévastateurs tels que la schizophrénie (SCZ), où l’étude de nouveaux gènes a servi de point de lancement pour de nouvelles lignes de recherche et d’interventionthérapeutique 5,6. Les gènes hébergeant le risque pour SCZ montrent l’expression biaisée dans le cortex préfrontal (PFC) pendant le développement prénatal et postnatal tôt, une région impliquée dans la pathologie de plusieurs désordres neuropsychiatriques7. En outre, les modèles de souris des désordres psychiatriques montrent l’activité anormale dans les réseauxde PFC 6,8,9. Ces résultats suggèrent que les gènes associés au SZC pourraient jouer un rôle dans le câblage développemental de cette région. Une étude plus approfondie utilisant des modèles animaux est nécessaire pour comprendre la contribution de ces gènes candidats à l’établissement de connexions dans le PFC et pour déterminer si ces gènes ont un rôle causal dans la pathogénie des troubles neuropsychiatriques. Les techniques de manipulation génétique chez la souris qui permettent l’étude des changements d’expression génétique sur des circuits neuronaux spécifiques pendant le développement prénatal et postnatal précoce sont une méthode prometteuse pour comprendre les mécanismes moléculaires qui relient les changements d’expression génétique au dysfonctionnement du PFC.
Les lignées génétiques de souris offrent une méthode pour étudier l’impact de gènes particuliers sur le développement et la fonction du cerveau. Cependant, s’appuyer sur des souris transgéniques peut être limitatif car il n’y a pas toujours de lignes disponibles dans le commerce pour examiner les effets de gènes spécifiques sur le développement de circuits neuronaux. En outre, il peut être extrêmement coûteux et long de développer des lignes de souris personnalisées. L’utilisation de stratégies intersectionnelles de manipulation génétique qui combinent des souris transgéniques avec des approches virales a révolutionnéla compréhension du cerveau 10,11,12. Malgré de nombreux progrès, les stratégies virales sont associées à certaines limitations selon le type de vecteur viral, y compris les limites de la capacité d’emballage qui peuvent restreindre l’expression virale13 et la toxicité cellulaire associée à l’expression virale14. En outre, dans la plupart des conditions expérimentales, l’expression génétique robuste utilisant le virus adéno-associé (AAV) exige approximativement 2 à 4 semaines15,rendant les stratégies virales courantes impossibles à manipuler des gènes pendant le développement postnatal tôt.
L’électroporation in utero (IUE) est une approche alternative qui permet un transfert de gènes rapide et peu coûteux16,17 qui, lorsqu’il est couplé avec l’étiquetage fluorescent et des approches pharmacogénétiques ou optogénétiques, fournit une plate-forme puissante pour disséquer la fonction des circuits neuronaux. En outre, avec le développement de CRISPR-Cas9 gènes d’édition du génome peuvent être surexprimés ou précisément modifiés par type de cellule spécifique knock-down ou knock-out de gènes spécifiques ou par la modulation des promoteurs18,19. Les approches de manipulation génétique utilisant l’IUE sont particulièrement avantageuses lorsque l’effet des gènes sur les circuits neuronaux doit être testé lors de fenêtres de développementétroites 20. L’IUE est une technique polyvalente et la surexpression peut être facilement accomplie en insérant un gène dans un vecteur d’expression sous un promoteur spécifique. Un contrôle supplémentaire de l’expression des gènes peut être réalisé en conduisant l’expression en utilisant des promoteurs de différentes forces ou en utilisant des promoteurs inductibles capables de contrôler temporellement l’expressiondes gènes 21,22. En outre, l’IUE permet le ciblage des cellules dans des couches corticales spécifiques, des types de cellules et des régions du cerveau, ce qui n’est pas toujours faisable enutilisant d’autres approches 5,17. Les progrès récents de la configuration IUE basés sur l’utilisation de trois électrodes, qui génère une distribution plus efficace des champs électriques, ont élargi le répertoire fonctionnel de cette méthode et permis aux scientifiques de cibler de nouveaux types de cellules et d’augmenter l’efficacité, la précision et le nombre de cellules qui peuvent êtreciblées 23,24. Cette technique a été récemment utilisée pour déterminer le rôle causal du composant de complément 4A (C4A), un gène lié à SCZ, dans la fonction PFC et la cognition précoce5.
Présenté ici est un pipeline expérimental qui combine des approches de transfert de gènes pour cibler de grandes populations de neurones excitatoires dans le cortex frontal, y compris le PFC, avec des paradigmes comportementaux qui permet non seulement l’étude des changements cellulaires et de niveau de circuit, mais permet également de surveiller le comportement tout au long du développement postnatal précoce et l’âge adulte. D’abord décrit est une méthode pour transfecter bilatéralement de grandes populations de couches (L) 2/3 neurones pyramidaux dans les régions corticales frontales. Ensuite, les tâches d’analyse du comportement social chez les souris juvéniles et adultes sont décrites. Le nombre de cellules peut être obtenu à la fin des tâches comportementales pour quantifier l’étendue et l’emplacement de la transfection cellulaire. En outre, le nombre de cellules transfectées peut être corrélé avec des données comportementales pour déterminer si un plus grand nombre de cellules transfectées conduit à de plus grandes perturbations du comportement.

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="663">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">13mm Silk Black Braided Suture</td>
			<td style="width:121px;">Havel&#39;s</td>
			<td style="width:161px;">SB77D</td>
			<td style="width:167px;">Suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11006-12</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">C270 Webcam</td>
			<td style="width:121px;">Logitech</td>
			<td style="width:161px;">N/A</td>
			<td>Record behavior</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Electroporator</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>See Figure 1 and 2 and Bullmann et al, 2015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps</td>
			<td>Bio Basic Inc.</td>
			<td style="width:161px;">BS466</td>
			<td>Pladmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fast Green FCF</td>
			<td style="width:121px;">Sigma</td>
			<td>F7252-5G</td>
			<td>Dye for DNA solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine scissors- sharp</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">14060-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand Gauze Sponges</td>
			<td>Fisher Scientific</td>
			<td>1376152</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gaymar Heating/Cooling</td>
			<td style="width:121px;">Braintree</td>
			<td style="width:161px;">TP-700</td>
			<td style="width:167px;">Heating Pad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipette puller</td>
			<td>Sutter Instrument,</td>
			<td style="width:161px;">P-97</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipettes</td>
			<td>Sutter Instrument,</td>
			<td>BF150-117-10</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hair Removal Lotion</td>
			<td style="width:121px;">Nair</td>
			<td style="width:161px;">N/A</td>
			<td>Hair removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">13002-10</td>
			<td>IUE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Open field maze- homemade acrylic arena</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>50 &#215; 50 &#215; 30 cm length-width-height</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">pCAG-GFP</td>
			<td style="width:121px;">Addgene</td>
			<td>11150</td>
			<td>Mammalian expression vector for expression of GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td style="width:161px;">N/A</td>
			<td>pressure injector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retractor - 2 Pronged Blunt</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">17023-13</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ring forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11103-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sterilizer, dry bead</td>
			<td style="width:121px;">Sigma</td>
			<td>Z378569</td>
			<td>sterelize surgical tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY</td>
			<td>Havel&#39;s</td>
			<td>HJ398</td>
			<td>Suture muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water bath</td>
			<td style="width:121px;">Cole-Parmer</td>
			<td>EW-12105-84</td>
			<td style="width:167px;">warming sterile saline</td>
		</tr>
	</tbody>
</table>

a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior

Alors que les études d’association à l’échelle du génome mettent en lumière les fondements génétiques hétérogènes de nombreuses maladies neurologiques, la nécessité d’étudier la contribution de gènes spécifiques au développement et à la fonction du cerveau augmente. S’appuyer sur des modèles muraux pour étudier le rôle de manipulations génétiques spécifiques n’est pas toujours faisable puisque les lignées de souris transgéniques sont assez coûteuses et que de nombreux nouveaux gènes associés à la maladie n’ont pas encore de lignées génétiques disponibles dans le commerce. En outre, il peut prendre des années de développement et de validation pour créer une ligne de souris. L’électroporation in utero offre une méthode relativement rapide et facile pour manipuler l’expression des gènes d’une manière spécifique de type cellulaire in vivo qui nécessite seulement le développement d’un plasmide d’ADN pour réaliser une manipulation génétique particulière. L’électroporation in utero bilatérale peut être utilisée pour cibler de grandes populations de neurones pyramidaux du cortex frontal. La combinaison de cette méthode de transfert de gènes avec des approches comportementales permet d’étudier les effets des manipulations génétiques sur la fonction des réseaux préfrontaux du cortex et le comportement social des souris juvéniles et adultes.

Développement et mise en œuvre réussis d’un électroporateur sur mesure et de trois électrodes à dents. Pour les IUI, un électroporateur sur mesure peu coûteux a été construit sur la base d’une conceptionprécédemment décrite 27 (figure 1A et figure 2). Une électrode à trois volets a étéfaite 23,24 à l’aide de forceps en plastique avec 2 ?...

Watch this Scientific Journal Video about A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior at JoVE.com

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

Le rôle des gènes récemment découverts associés à la maladie dans la pathogénie des désordres neuropsychiatriques demeure obscur. Une technique bilatérale modifiée d’électroporation in utero permet le transfert de gènes dans de grandes populations de neurones et l’examen des effets causatifs des changements d’expression génétique sur le comportement social.

Un pipeline utilisant l’électroporation bilatérale in utero pour interroger les influences génétiques sur le comportement des rongeurs

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

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Biology

3.9K vues.  Boston University. Le rôle des gènes récemment découverts associés à la maladie dans la pathogénie des désordres neuropsychiatriques demeure obscur. Une technique bilatérale modifiée d’électroporation in utero permet le transfert de gènes dans de grandes populations de neurones et l’examen des effets causatifs des changements d’expression génétique sur le comportement social.

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

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

En injection intraventriculaire Utero et l&#39;électroporation d&#39;embryons de rat E16

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

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Biologie

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

En injection intraventriculaire Utero et l&#39;électroporation d&#39;embryons de souris E15

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Méthode pour la culture d&#39;embryons de poulet Early ex vivo (Nouvelle Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Mont entier hybridation in situ d&#39;embryons de poulet début

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Méthode de coloration Mont anticorps entier de Chick

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Dosage de l&#39;induction neurale dans l&#39;embryon de poulet

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

En électroporation in vivo et ex utero pour l&#39;expression génique dans les cellules ganglionnaires de la rétine de la souris

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Les études génétiques des protéines humaines réparation de l&#39;ADN utilisant de la levure comme système modèle

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Mesure et analyse de la production extracellulaire acide pour Déterminer glycolytique Taux

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...