We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.

NOT: canlı hayvanlar kullanarak tüm protokolleri gözden ve Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmış ve resmi laboratuvar hayvanlarının bakımı ve kullanımı için yöntemler onaylı takip gerekir gerekir. Bütün tahlil temiz bir laminer akış tezgahı üzerinde, steril tekniği kullanılarak yapılmalıdır. Eldiven ve maske, istenirse, bu işlem sırasında giyilmelidir. Embriyonik Fare İç Kulak 1. Diseksiyon <ol><li> Corti eksplant kültürlerin organ için ayarlama: <ol><li> Yaklaşık 30 dakika, UV ışık yakarak laminer akış doku kültürü kapağı sterilize ve kullanımdan önce% 70 etanol ile bir yüzeyin dezenfekte edilmesi. Buna ek olarak, ince diseksiyon araçları ve otoklav yoluyla embriyo diseksiyon veya kullanımdan önce en az 20 dakika süreyle% 70 etanol iliklerine Sylgard kaplı yemekleri dahil olmak üzere tüm diseksiyon aletleri sterilize. Temiz bir laminer akış clea hava kurumaya yemekleri ve araçlar% 70 etanol dökün ve izinn tezgah kullanmadan önce. </li><li> Hazırlama steril Hank Dengeli Tuz Çözeltisi (HBSS) / (4- (2-hidroksietil) -1-piperazinetansülfonik asit), HEPES 10x HBSS ve% 0.5% 10 eklenmesi ve yaklaşık pH&#39;ı 7.2&#39;ye ayarlayın ve filtre sterilizasyonu ile (HEPES) karışımı 4 ° C&#39;de, nihai çözelti saklayın. Daha iyi prosedür boyunca doku korumak için soğutulmuş HBSS / hepes çözeltisi içinde tüm diseksiyonları uygulayın. </li><li> 15 ml&#39;lik steril bir polipropilen tüp içinde taban membran matrisi bir 300 ul&#39;lik bölüntü, steril (4 ° C) soğuk Dulbecco Modifiye Edilmiş Eagle Ortamı (DMEM) 5 ml ilave etmek suretiyle taban membran matrisi ile kaplayın cam alt yemekler. Içeriğini vorteks ile karıştırın ve kasesinin ortasına kadar mevcut tüm kültür kapsar, böylece her bir kültür kabı merkezine bazal membran matris DMEM karışımından 150 ul ilave edin. Kullanımdan önce en az 45 dakika süre ile 37 ° C inkübatör yemekleri ve mağaza örtün. </li><li> Th içine HBSS soğutulmuş 4-5 ml dökünSteril polistiren petri kapları REE ve kaput kapalı bırakın. </li><li> DMEM 9 ml N2 takviyeleri 100 ul, 10 ug / ml Cipro ve FBS, 1 ml ilave edilerek steril bir 15 ml&#39;lik polipropilen bir tüp içinde kültür ortamı hazırlanır. </li></ol></li><li> Embriyoların izole edilmesi: NOT: embriyonik farelerden alınan koklear eksplantlarm Bu prosedürde, hasat iç kulaklar embriyonik gün (E) den gübreleme gün sonra 13 temporal kemik E1 9 olarak kabul ediliyor. protokol de E12 dan E18 başlayan embriyonik fare iç kulakları ile kullanılabilir. <ol><li> CO 2 ile zamanlanmış gebe fare Euthanize ve servikal dislokasyon veya uygun onaylanmış protokoller ile hayvan kurban. Doku kültürü odasında sterilite korumak için uzak laminer akış doku kültürü kaputu gelen euthanization yürütmek. </li><li> Yukarı bakacak şekilde karın kağıt havlu üzerine ötenazi fare koyun ve% 70 etanol ile karın dezenfekte edin. </li><li> Cildi kapma karın boşluğu açınBaşka bir el kullanılarak makas ile orta hat boyunca kavisli bir eliyle forseps ve kesme epidermis ve kas ile. Her iki tarafta iki dik kesim yapmak ve forseps ile bilateral uterin boynuz kaldırarak dikkatle rahim çekin ve bağ dokusu kullanılarak makas altından ayırmak. </li><li> Laminer akışlı temiz bir tezgah soğutulmuş diseksiyon çözeltisi (HBSS / hepes karışımı) ve transfer ihtiva eden bir petri tabağına embriyonik zinciri yerleştirin. </li><li> Plasenta dikkatlice embriyolar çıkarın ve diseksiyon çözeltisi içeren steril Petri kapları birine yerleştirin. Çözüm kanlı dönerse, yeni bir Petri kabı taşıyabilirsiniz. NOT: Bu noktada, embriyolar, bir Theiler evreleme kılavuzunu kullanarak sahnelenecek edilebilir. </li><li> Temiz forseps veya makasla ile boyun bölgesinde kısma embriyolar başını kesmek ve soğutulmuş diseksiyon çözeltisi içeren taze Petri kabındaki başkanları yerleştirin. </li></ol></li><li> İç kulak Diseksiyon: <ol><li> Yeri oSoğuk diseksiyon içeren bir steril sylgard çanak ne embriyo baş </li><li> Çözelti. ~ 1.6x bir büyütmede bir mikroskop altında çalışarak, göz bölgesi (Şekil 1A) etrafında ya da daha yakın Minutien işaretçilerini yerleştirerek embriyo baş hareketsiz. </li><li> Steril forseps iki çift kullanarak dikkatli bir şekilde cilt kaldırmak ve orta hat (Şekil 1B) boyunca sırt tarafında kafatasını açmak. Kranial kavite ve temporal kemiklerin içinde yer alan iç kulaklar, bu aşamada (Şekil 1C, D) tespit edilebilir beyni çıkarın. Genellikle, iç kulak çevresine mevcut kan damarı, astar, bu aşamada (Şekil 1D), iç kulak belirlemek için bir işaret olarak kullanılabilir. </li><li> Dokuda altına forseps yerleştirerek ve soğuk diseksiyon çözüm içeren yeni bir çanak kafatası ve transfer tabanından iç kulak izole ederek zamansal kemiklerden iç kulak incelemek (Şekil 1E, F). </li></ol></li></ol> Corti Eksplant Kültürler Organ 2. Nesil NOT: Bu gelişme aşamasında, doku kıkırdak ve kolayca forseps kullanılarak disseke edilebilir. <ol><li> Orient ventral yüzü (Şekil 2A) yukarı bakacak ve yavaşça iç kulak (Şekil 2B) vestibüler bölümü ile Minutien pim keskin ucunu takarak iç kulak stabilize böylece iç kulak. </li><li> Kullanımı ultra ince forseps koklea (Şekil 2C) kıkırdak kaldırmak dikkatlice oval pencereye yakın forseps bir ucunu kullanarak ve bir kesi yaparak örten kıkırdak kesip. NOT: Bu örten kıkırdak bazen altına kazıma ile altta yatan koklear kanal gelen kıkırdak serbest bırakmak için kapalı forseps kullanmak yararlı olduğu durumda koklear kanal ile kaynaşmış olarak forseps kıkırdak içine çok derin takılı olmadığından emin olmak için önemlidir sKıkırdak, sert bir yere. Bu gelişme aşamasında, koklear spiral uzunluğu bir dönüş sadece dörtte üçü olduğunu. </li><li> Sonraki baz ya da çok apeks de ya koklear kanal tercih edilen bölgede forseps koyarak duyusal epitel maruz ve yavaşça koklea (Şekil 2B) çatısı çekerek. NOT: Kokleanın çatı o analizini engelleyen duyusal epitel maske gibi tamamen kaldırılmalıdır. NOT: duyusal epitel gözyaşı kolay olduğu gibi bu işlem nazikçe yapılmalıdır. </li><li> Koklear eksplant tabanı eşit düz böylece son adım olarak, dikkatle maruz duyusal epitel yatan bağ doku kaldırmak. </li><li> Forseps kullanarak iç kulak vestibüler kısmından disseke koklea izole edin. Bu, daha önce ya da aşama 2.4 (Şekil 2E) sonra gerçekleştirilebilir. </li><li> Bir bodrum membra içine disseke koklear duyusal epitel aktarınne matris kaplı kültür de bir 1,5 mm steril scooper (Şekil 2F) kullanılarak. </li><li> Orient epitel lümenal yüzeyi ile koklear eksplant yukarı bakacak ve dikkatle doku dümdüz ve hafifçe çanak ekleyerek taze kültür ortamı 150 ul ile değiştirmek için bazal membran matris-DMEM çözüm aspire. Bakım, her eksplant kaplamalı cam lamel iyi yapışır ve kültür ortamı içinde yüzen olmadığından emin olmak için alınmalıdır. Koklear eksplant zarar vermemek için cam kuyuya yapıştırılması sırasında forseps ucu kadar sivri olduğundan emin olun. </li><li> Yavaşça İstenilen bir zaman, in vitro olarak, genellikle 3-6 gün (DIV),% 5 CO2 ile 37 ° C &#39;de, bir doku kültürü kuluçka makinesi içinde bir steril 150 mm kültür çanağı ve yerine kültür kaplarına transfer. 1 DIV sonra, koklear eksplant kültürü çanak iyi yapışmış emin olmak için bir mikroskop altında kültürleri incelemek. </li><li> Sonrasıistenen zaman uzunlukları için in vitro cubation, kültürler imünohistokimya için işlenir. Not: eksplant kültürleri genellikle gelişim aşamasına, P0 eşdeğerdir 6 DIV için inkübe edilir. In vitro inkübasyondan sonra, kültürler, immünohistokimya gibi, daha aşağıda uygulama (Şekil 3B) için işlenebilir, RT-PCR, in situ hibridizasyon ve / veya Western blot. </li></ol> 3. Elektroporasyon aracılı gen transferi <ol><li> Elektroporasyon için ayarlama: <ol><li> Otoklavda veya yaklaşık 20 dakika boyunca% 70 etanol içinde ıslatılarak bir 100 mm Sylgard kaplı cam tabak sterilize ve kullanımdan önce temiz bir tezgah içinde kurumaya bırakın. </li><li> Uygun maksi veya Midi-prep kitleri kullanılarak seçim bir ifade vektörü DNA hazırlayın. Bu protokolde kullanılan ifade vektörleri pIRES2-Atoh1.EGFP ve pCLIG-NeuroD1.EGFP vardır. DNA nihai konsantrasyon steril DNA, en az 1 ug / ul olmalıdır /RNAse içermeyen su. </li></ol></li><li> Embriyonik koklear eksplantlarından içine DNA electroporating: <ol><li> Taze 100 mm Sylgard kaplı çanak plazmid DNA çözeltisinin 10 ul ekle ve DNA çözeltisi (adım 2.5 de elde edilmiş), tam kesilmiş koklear eksplant aktarın. </li><li> Çanak düzlemine dikey olacak şekilde yukarı bakacak şekilde epitelyum lümenal yüzeyi ile hafifçe koklea yatırın. </li><li> Duyusal epiteli ve koklea tabanına bitişik bulunan pozitif raket (anot) doğru negatif kürek (katot) ile koklea iki tarafında elektrot kürekler yerleştirin. Not: DNA, negatif yüklü olduğu için, pozitif elektrot ve duyu epitel bitişik sonda katot ucuna yerleştirilerek negatiften göç; DNA, çoğunlukla duyusal epitel yüzeyine geçer. </li><li> Elektro-kullanma, 24 mV 9-10 darbeler teslim, t ayak pedalı ile 30 msn darbe süresiO Electroporator ve ardından Electroporated cochlea&#39;ya sıcak kültür ortamı 100 ul ekleyin. </li><li> Tekrar tüm koklear eksplant ve ilgi tüm genlerin DNA için 3.2.1-4 adımları ve (adım 2.7) kaplama için bazal membran matris kaplı çanak içine electroporated cochleae aktarın. Daha sonra, bağışıklık kimyası için işlenecektir arzu edilen bir zaman uzunlukları için 37 ° C,% 5 CO2 ile nemlendirilmiş inkübatörde kültür Electroporated cochleae. NOT: CD1 fare (yaklaşık 8-12 yavru) bütün bir çöp gelen koklea, izole microdissected, Electroporated ve &lt;4 saat içinde kaplama olabilir. </li></ol></li></ol> 4. Analiz Koklear Eksplantın Kültürlerin Not: kültürleri genellikle gelişim aşamasına, P0 eşdeğerdir 6 DIV için inkübe edilir. In vitro inkübasyondan sonra, kültürler sabitlenmiş ve immünolojik hücre kimyası için işlendi. <ol><li> Inc istenilen uzunlukta sonraubation, kültür ortamı çıkarın ve hızlı bir şekilde fosfat-tamponlu salin (PBS) &#39;de koklear eksplant yıkayın ve oda sıcaklığında 15 dakika boyunca (PBS içerisinde hazırlanmıştır)% 4 paraformaldehid içinde inkübe edilir. </li><li> 15 dakika boyunca üç kez ekleyerek PBS ile fiksatif yıkayın ve PBS içinde durulayın. </li><li> % 10 keçi serumu içeren PBS-T ile bloke önce 30 dakika için PBS-T (PBS +% 0.5 Tween) ile geçirgenliği. </li><li> 4 ° C de bir gece boyunca,% 1 keçi serumu ile PBS-T ile primer antikor ihtiva eden birincil antikor çözelti içinde inkübe edilir. </li><li> Uygun Alexa-konjuge sekonder antikor ilave edilmeden önce PBS-T her biri 15 dakika kullanılarak oda sıcaklığında koklea üç kez yıkayın. </li><li> Koklear eksplant cam lamel kültüre olduğundan, çanak lamel izole ve bir slayt monte edilerek tek tek ele alınabilir. Bu, oda sıcaklığında en az 1 saat için çözücü, OS30 kültür çanağı ıslatılmasıyla elde edilebilir. Steril jilet kullanarak, d lamel ayırmakish ve bir slayt monte ve bir floresan mikroskop altında görselleştirmek. </li></ol>

<ol>
	<li>Sobkowicz, H. M., Bereman, B., Rose, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+development+of+the+organ+of+Corti+in+culture.">Organotypic development of the organ of Corti in culture.</a> J Neurocytol. 4 (5), 543-572 (1975).</li><li>Zheng, J. L., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Math1+induces+robust+production+of+extra+hair+cells+in+postnatal+rat+inner+ears.">Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears.</a> Nat Neurosci. 3 (6), 580-586 (2000).</li><li>Zheng, J. L., Shou, J., Guillemot, F., Kageyama, R., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes1+is+a+negative+regulator+of+inner+ear+hair+cell+differentiation.">Hes1 is a negative regulator of inner ear hair cell differentiation.</a> Development. 127 (21), 4551-4560 (2000).</li><li>Woods, C., Montcouquiol, M., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+regulates+development+of+the+sensory+epithelium+in+the+mammalian+cochlea.">Math1 regulates development of the sensory epithelium in the mammalian cochlea.</a> Nat Neurosci. 7 (12), 1310-1318 (2004).</li><li>Jones, J. M., Montcouquiol, M., Dabdoub, A., Woods, C., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+differentiation+and+DNA+binding+(Ids)+regulate+Math1+and+hair+cell+formation+during+the+development+of+the+organ+of+Corti.">Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti.</a> J Neurosci. 26 (2), 550-558 (2006).</li><li>Dabdoub, 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=Sox2+signaling+in+prosensory+domain+specification+and+subsequent+hair+cell+differentiation+in+the+developing+cochlea.">Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (47), 18396-18401 (2008).</li><li>Doetzlhofer, A., Basch, M. L., Ohyama, T., Gessler, M., Groves, A. K., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hey2+regulation+by+FGF+provides+a+Notch-independent+mechanism+of+maintaining+pillar+cell+fate+in+the+organ+of+Corti.">Hey2 regulation by FGF provides a Notch-independent mechanism of maintaining pillar cell fate in the organ of Corti.</a> Dev Cell. 16 (1), 58-69 (2009).</li><li>Puligilla, C., Dabdoub, A., Brenowitz, S. D., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2+induces+neuronal+formation+in+the+developing+cochlea.">Sox2 induces neuronal formation in the developing cochlea.</a> J Neurosci. 20 (2), 714-722 (2010).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1995).</li><li>Sher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+and+postnatal+development+of+the+inner+ear+of+the+mouse.">The embryonic and postnatal development of the inner ear of the mouse.</a> Acta Otolaryngol Suppl. 285, 1-77 (1971).</li><li>Torres, M., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+vertebrate+inner+ear.">The development of the vertebrate inner ear.</a> Mech Dev. 71 (1-2), 5-21 (1998).</li><li>Brown, S. T., Martin, K., Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+inner+ear+induction.">Molecular basis of inner ear induction.</a> Curr Top Dev Biol. 57, 115-119 (2003).</li><li>Puligilla, C., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+world&#8217;s+best+hearing+aid;+regulation+of+cell+fate+in+the+cochlea.">Building the world&#8217;s best hearing aid; regulation of cell fate in the cochlea.</a> Curr Opin Genet Dev. 19 (4), 368-373 (2009).</li><li>Wu, D. K., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+inner+ear+development.">Molecular mechanisms of inner ear development.</a> Cold Spring Harb Perspect Biol. 4 (8), a008409 (2012).</li><li>Holt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral-mediated+gene+transfer+to+study+the+molecular+physiology+of+the+mammalian+inner+ear.">Viral-mediated gene transfer to study the molecular physiology of the mammalian inner ear.</a> Audiol Neurootol. 7 (3), 157-160 (2002).</li><li>Stone, I. M., Lurie, D. I., Kelley, M. W., Poulsen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+to+hair+cells+and+support+cells+of+the+murine+cochlea.">Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea.</a> Mol Ther. 11 (6), 843-848 (2005).</li></ol>

No conflicts of interest declared.

Dahil olmak üzere, bir fare, iç kulağın membranöz labirent içindeki tüm hücreler duyusal duyunun ve spiral gangliyon nöronlarının her E8 10-14 civarında, ektodermin Beyin bitişiğinde yer otocyst placodally türevi elde edilir. E11 içinde otocyst ventral bölgesi koklear kanal oluşturmak üzere uzanır ve geliştirme devam ederken haline koklea içindeki epitel hücreleri yanı sıra otocyst diğer bölgelerinde bir grup, daha sonra ortaya çıkaracaktır prosensory yamalar olarak belirtilen me...

The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis. 
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results. 
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HBSS</td>
			<td style="width:192px;">Gibco</td>
			<td style="width:161px;">14065-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">HEPES</td>
			<td>Gibco</td>
			<td style="width:161px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Dulbecco&#8217;s Modified Medium</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">12430-054</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Fetal Bovine Serum</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10082</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">N-2 Supplement (100X)</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Ciprofloxacin Hydrochloride</td>
			<td dir="LTR" style="width:192px;">Cellgro</td>
			<td dir="LTR" style="width:161px;">61-277-RF</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Glass Dish 60mm</td>
			<td dir="LTR" style="width:192px;">Kimble Chase</td>
			<td dir="LTR" style="width:161px;">23062-6015/23064-6015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Glass Dish 100 mm</td>
			<td dir="LTR">Kimble Chase</td>
			<td style="width:161px;">23064-10015/23062-10015</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Minutien Pins</td>
			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
			<td style="width:161px;">26002-15</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Dumont # 5 Forceps</td>
			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
			<td dir="LTR" style="width:161px;">11251-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Pulse Generator&#160;</td>
			<td dir="LTR">Protech International Inc</td>
			<td style="width:161px;">CUY21Vivo-SQ</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Glass Bottom Culture Dishes</td>
			<td dir="LTR" style="width:192px;">MatTek</td>
			<td dir="LTR" style="width:161px;">P35G-0-10-C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Matrigel Matrix</td>
			<td dir="LTR" style="width:192px;">BD Biosciences</td>
			<td dir="LTR" style="width:161px;">356237</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Culture Dish, 60 X 15 mm</td>
			<td dir="LTR" style="width:192px;">Becton Dickinson</td>
			<td style="width:161px;">353037</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Tissue&#160; Culture Dishes</td>
			<td dir="LTR" style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">639160</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Phosphate Buffered Saline</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">OS-30</td>
			<td dir="LTR" style="width:192px;">Dow Corning</td>
			<td dir="LTR" style="width:161px;">4021768</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Fluoromount</td>
			<td dir="LTR" style="width:192px;">Southern Biotech</td>
			<td dir="LTR" style="width:161px;">0100-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Conical Tubes, 15ml</td>
			<td style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">188261</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Myosin 6</td>
			<td>Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6791</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Myosin 7a</td>
			<td style="width:192px;">Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6790</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">TuJ1</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:161px;">T2200</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of embryonic mouse cochlear explants and gene transfer by electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest...

Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Elektroporasyon tarafından Embriyonik Fare Koklear Eksplantlarından Kültür ve Gen Transferi

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

culture-embryonic-mouse-cochlear-explants-gene-transfer

Research

JoVE Journal

Developmental Biology

12.8K Görüntüleme.  Medical University of South Carolina, College of Medicine. We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development. 

Video: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Primer Hücre Kültürü Araştırmaları Chick retina Verimli Gen Transferi: Bir<em&gt; Eski-ovo</em&gt; Elektroporasyon Yaklaşımı

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Gelişim Biyolojisi

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Kültür ve Co-Kültür Mouse Yumurtalıklar ve Yumurtalık Foliküller

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

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.

Fare Hipokampal Doku Ex Utero Elektroporasyon ve Organotipik Dilim Kültür

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

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Serum serbest Tek tabakalı Kültüründe Fare Embriyonik Kök Hücre Sinir Farklılaşma

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

Elektroporasyon ve Kimyasal Reaktif tedaviler için Tavuk Embriyolar gelen bütün Retina Eksplantlarındaki

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Tavuk İşitsel Organ gen Transferi tarafından<em&gt; Ovo olarak</em&gt; Mikro-elektroporasyon

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Multiphoton Mikroskopi Canlı hücre Görüntüleme için Yetişkin Transgenik Zebra balığı Retina Eksplantlarından Kültür

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Fare Embriyonik Böbreklerinin Diseksiyonu ve Kültürü

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

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.

CRISPR-aracılı kaybı serebellar granül işlev analizinin gen transferi Elektroporasyon tabanlı rahim içinde kullanarak hücreleri

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

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Fare İskelet Kas Ekstranında Primer Miyoblastların İzolasyon ve Farklılaşması

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...