Lisa Kretsge'ye eleştirel geri bildirim ve makaleyi düzenlediği için teşekkür ederiz. Cruz-Martín laboratuvarındaki tüm araştırma asistanlarına teşekkür ederiz. Tripolar elektrodun tasarımına giriş için Andrzej Cwetsch'e ve konfokal mikroskobun kullanımı için Todd Blute ve Boston Üniversitesi Biyoloji Görüntüleme Çekirdeği'ne teşekkür ederiz. Bu çalışma bir NARSAD Genç Araştırmacı Hibesi (AC-M, #27202), Brenton R. Lutz Ödülü (ALC), I. Alden Macchi Ödülü (ALC), NSF NRT UtB: Neurophotonics Ulusal Araştırma Bursu (ALC, #DGE1633516) ve Boston Üniversitesi Lisans Araştırma Fırsatları Programı (WWY) tarafından desteklendi. Fon sağlayıcılar çalışma tasarımı, veri toplama ve analiz, yayınlama kararı veya makalenin hazırlanmasında hiçbir rol oynamamışlardır.

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation.">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> Journal of Neuroscience Methods. 143 (2), 151-158 (2005).</li><li>Soma, 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=Development+of+the+mouse+amygdala+as+revealed+by+enhanced+green+fluorescent+protein+gene+transfer+by+means+of+in+utero+electroporation.">Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.</a> Journal of Comparative Neurology. 513 (1), 113-128 (2009).</li></ol>

Yazarlar rakip çıkarlar beyan etmemektedir.

Burada, frontal kortikal nöronların büyük popülasyonlarında ilgi çekici yeni genlerin manipülasyonu ile farelerdeki davranışsal tahlilleri birleştiren bir boru hattı açıklanmaktadır. Ayrıca, bu boru hattı hem erken doğum sonrası gelişim sırasında hem de yetişkinlikte aynı farelerdeki davranışların uzunlamasına incelenmesine izin verir. Bu teknik, zaman ve masraflar açısından maliyetli olabilecek genetik hayvan modellerine güvenme ihtiyacını atlar. Bu protokolün gücü, son GWAS'ın yeni...

Genom çapındaki ilişki çalışmaları (GWAS), beyin patolojileri 1 ,2,3,4ile ilişkili yeni aday genlerin keşfini yönlendirmektedir. Bu çalışmalar, yeni genlerin araştırılmasının yeni araştırma ve terapötik müdahale hatları için bir fırlatma noktası olarak hizmet ettiği şizofreni (SCZ) gibi yıkıcı nöropsikiyatrik bozuklukları anlamada özellikle yararlı olmuştur5,6. SCZ için risk barındıran genler, doğum öncesi ve erken postnatal gelişim sırasında prefrontal kortekste (PFC) önyargılı ifade gösterir, çeşitli nöropsikiyatrik bozuklukların patolojisine karışan bir bölge7. Ek olarak, psikiyatrik bozuklukların fare modelleri PFC ağlarında anormal aktivite sergiler6,8,9. Bu sonuçlar, SZC ile ilişkili genlerin bu bölgenin gelişimsel kablolarında rol oynayabileceğini göstermektedir. Bu aday genlerin PFC'de bağlantıların kurulmasına katkısını anlamak ve bu genlerin nöropsikiyatrik bozuklukların patogenezinde nedensel bir rolü olup olmadığını belirlemek için hayvan modelleri kullanılarak daha fazla araştırma yapılması gerekmektedir. Farelerde, doğum öncesi ve erken postnatal gelişim sırasında belirli nöronal devrelerdeki gen ekspresyon değişikliklerinin incelenmesine izin veren genetik manipülasyon teknikleri, gen ekspresyon değişikliklerini PFC disfonksiyonu ile ilişkilendiren moleküler mekanizmaları anlamak için umut verici bir yöntemdir.
Genetik fare çizgileri, belirli genlerin beyin gelişimi ve işlevi üzerindeki etkisini incelemek için bir yöntem sunar. Bununla birlikte, transgenik farelere güvenmek sınırlayıcı olabilir, çünkü belirli genlerin sinir devreleri geliştirme üzerindeki etkilerini incelemek için her zaman ticari olarak kullanılabilir çizgiler yoktur. Ayrıca, özel fare çizgileri geliştirmek son derece maliyetli ve zaman alıcı olabilir. Transgenik fareleri viral yaklaşımlarla birleştiren kesişimsel genetik manipülasyon stratejilerinin kullanılması, beynin anlaşılmasında devrim yaptı10,11,12. Çok ilerlemeye rağmen, viral stratejiler viral vektör tipine bağlı olarak, viral ifade13'ü ve viral ifade ile ilişkili hücre toksisitesini kısıtlayabilecek ambalaj kapasitesi sınırları da dahil olmak üzere belirli sınırlamalarla birlikte gelir14. Ayrıca, çoğu deneysel durumda, adeno ilişkili virüs (AAVs) kullanarak sağlam gen ekspresyonu yaklaşık 2 ila 4 haftagerektirir 15, erken doğum sonrası gelişim sırasında genleri manipüle etmek için rutin viral stratejileri imkansız hale getirir.
Rahim elektroporasyonunda (UUİ), floresan etiketleme ve farmakogenetik veya optogenetik yaklaşımlarla birleştiğinde nöronal devrelerin işlevini parçalamak için güçlü bir platform sağlayan hızlı ve ucuz bir gen transferi sağlayan alternatif bir yaklaşımdır16,17. Ek olarak, CRISPR-Cas9 genom düzenleme genlerinin geliştirilmesiyle, hücre tipi spesifik devirme veya belirli genlerin nakavt edilmesi veya uyarıcıların modülasyonu yoluyla aşırı ifade edilebilir veya tam olarak değiştirilebilir18,19. İEÜ'leri kullanan gen manipülasyon yaklaşımları özellikle genlerin nöronal devreler üzerindeki etkisinin dar gelişimsel pencereler sırasında test edilmesi gerektiğinde avantajlıdır20. IUE çok yönlü bir tekniktir ve aşırı ifade, belirli bir promotör altında bir ifade vektörüne bir gen eklenerek kolayca gerçekleştirilebilir. Gen ekspresyonunun ek kontrolü, farklı güçlerdeki promotörler kullanılarak veya gen ekspresyonunun zamansal olarak kontrol edilebilen indüklenebilir promotörleri kullanarak ifadeyi yönlendirerek elde edilebilir21,22. Ayrıca, İEÜ, belirli kortikal katmanlar, hücre tipleri ve beyin bölgelerindeki hücrelerin hedeflanmasına izin verir, bu da diğer yaklaşımlar5,17kullanılarak her zaman mümkün değildir. Daha verimli bir elektrik alanı dağılımı oluşturan üç elektrot kullanımına dayanan UUİ yapılandırmasındaki son gelişmeler, bu yöntemin işlevsel repertuarını genişletti ve bilim adamlarının yeni hücre türlerini hedeflemelerine ve hedeflenebilecek hücre verimliliğini, doğruluğunu ve sayısını artırmalarına izin verdi23,24. Bu teknik son zamanlarda PFC fonksiyonunda ve erken biliş5'teSCZ'ye bağlı bir gen olan kompleman bileşeni 4A'nın (C4A)nedensel rolünü belirlemek için kullanılmıştır.
Burada sunulan, PFC de dahil olmak üzere frontal korteksteki büyük uyarıcı nöron popülasyonlarını hedeflemek için gen transferi yaklaşımlarını, sadece hücre ve devre düzeyindeki değişikliklerin incelenmesini sağlamakla kalmayıp, aynı zamanda davranışın erken doğum sonrası gelişim ve yetişkinlik boyunca izlenmesini sağlayan davranış paradigmalarıyla birleştiren deneysel bir boru hattıdır. İlk olarak, frontal kortikal bölgelerdeki büyük tabaka (L) 2/3 piramidal nöron popülasyonlarını iki taraflı olarak transfect etmek için bir yöntemdir. Daha sonra, genç ve yetişkin farelerde sosyal davranışları test etme görevleri özetlenmiştir. Hücre transfeksiyonunun kapsamını ve yerini ölçmek için davranışsal görevlerin tamamlanmasıyla hücre sayıları elde edilebilir. Ayrıca, transfected hücre sayısı, daha fazla sayıda transfected hücrenin davranışta daha fazla pertürbasyona yol açıp yol açtığını belirlemek için davranışsal verilerle ilişkilendirilebilir.

<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

Genom çapındaki ilişki çalışmaları birçok nörolojik hastalığın heterojen genetik temellerine ışık tuttukça, spesifik genlerin beyin gelişimine ve işlevine katkısını inceleme ihtiyacı artmaktadır. Belirli genetik manipülasyonların rolünü incelemek için fare modellerine güvenmek her zaman mümkün değildir, çünkü transgenik fare çizgileri oldukça maliyetlidir ve birçok yeni hastalıkla ilişkili gen henüz ticari olarak mevcut genetik çizgilere sahip değildir. Ayrıca, bir fare satırı oluşturmak için geliştirme ve doğrulama yıllar alabilir. Rahim elektroporasyonunda, gen ekspresyonunu hücre tipi spesifik bir şekilde manipüle etmek için nispeten hızlı ve kolay bir yöntem sunar in vivo, sadece belirli bir genetik manipülasyonu elde etmek için bir DNA plazmidi geliştirmeyi gerektirir. Utero elektroporasyonda bilateral, frontal korteks piramidal nöronların büyük popülasyonlarını hedeflemek için kullanılabilir. Bu gen transfer yöntemini davranışsal yaklaşımlarla birleştirmek, genetik manipülasyonların prefrontal korteks ağlarının işlevi üzerindeki etkilerini ve genç ve yetişkin farelerin sosyal davranışlarını incelemenizi sağlar.

Özel yapım bir elektroporatör ve üç uç elektrotunun başarılı gelişimi ve uygulanması. İEÜ'ler için, daha önce tanımlanmış bir tasarım27'ye (Şekil 1A ve Şekil2) dayanan ucuz bir özel yapım elektroporatör inşa edildi. Uçlarına bağlı2negatif elektrotlu plastik tokmaklar kullanılarak23 ,24 prong elektrodu yapıldı ve pozitif elektrot ...

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

Nöropsikiyatrik bozuklukların patogenezinde yakın zamanda keşfedilen hastalıkla ilişkili genlerin rolü belirsizliğini korumaktadır. Modifiye edilmiş bir in utero elektroporasyon tekniği, büyük nöron popülasyonlarında gen transferine ve gen ekspresyon değişikliklerinin sosyal davranış üzerindeki nedensel etkilerinin incelenmesine izin verir.

Kemirgen Davranışı Üzerindeki Genetik Etkileri Sorgulamak için İkili İn Utero Elektroporasyonunu Kullanan Bir Boru Hattı

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

a-pipeline-using-bilateral-utero-electroporation-to-interrogate

Research

JoVE Journal

Biology

3.9K Görüntüleme.  Boston University. Nöropsikiyatrik bozuklukların patogenezinde yakın zamanda keşfedilen hastalıkla ilişkili genlerin rolü belirsizliğini korumaktadır. Modifiye edilmiş bir in utero elektroporasyon tekniği, büyük nöron popülasyonlarında gen transferine ve gen ekspresyon değişikliklerinin sosyal davranış üzerindeki nedensel etkilerinin incelenmesine izin verir.

Video: 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.

E16 Rat Embriyolar Utero İntraventriküler Enjeksiyon ve Elektroporasyon

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

Biyoloji

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. 

E15 Fare Embriyolar Utero İntraventriküler Enjeksiyon ve Elektroporasyon

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.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

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.

Erken Civciv Embriyolar in situ Hibridizasyon Çift Tüm Dağı

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.

Chick Tüm Dağı Antikor Boyama Yöntemi

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.

Civciv Embriyo Sinir İndüksiyon için Testi

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.

Fare Retina Ganglion Hücreleri Gen İfadesi utero ve ex vivo Elektroporasyon

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.

İnsan DNA Onarım Proteinler Genetik Araştırmalar Maya Model Sistemi olarak kullanma

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.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

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.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

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