The authors were supported by: a CancerFree KIDS Research Grant, a March of Dimes Basil O&#8217;Connor Starter Scholar Award (#5-FY14-32), a Cincinnati Children&#8217;s Hospital Medical Center (CCHMC) Trustee Grant Award; a CCHMC Research Innovation and Pilot Funding Award; and CCHMC developmental funds. Authors also acknowledge the Capel laboratory for the initial optimization of this technique.

在这些研究中使用的所有小鼠是从Charles River实验室得到的CD-1小鼠。以前培养实验也已在其它菌株，如C57BL / 6J（数据未显示）进行的，但任何应变都可以使用。怀孕的成年女性约为2-3个月之久，并通过CO 2吸入颈椎脱位和前胚胎切除双侧开胸进行安乐死。小鼠饲养按照美国国立卫生研究院的指导方针，以及实验方案被批准由辛辛那提儿童医院医学中心的机构动物护理和使用委员会。 1.准备仪器仪表，文化传媒，和菜名<ol><li>制备一个70％的乙醇溶液。高压灭菌去离子水（用于制作加湿室以及制造/稀释溶液）。通过喷雾用70％的乙醇消毒清扫工具（钳子，剪刀，和27G的针头注射器）。 </li><li>备含有的1M钙和镁（80克NaCl，2克氯化钾，14.4克的 Na 2 HPO 4，2.4克KH 2 PO 4，6.75 ml的氯化钙 ，3.75毫升的1M MgCl 2的10X磷酸盐缓冲盐水（PBS） ）。搅拌使之溶解在1升无菌高压灭菌的水，然后过滤用滤纸。稀释10倍的水，使1×PBS。 </li><li>热灭活的胎牛血清（FBS），在55℃进行30分钟，并过滤消毒用0.22μm注射器过滤器。 </li><li>准备38个ml的1X完全培养基（称为完全DMEM，cDMEM），由Dulbecco改良的Eagle培养基（DMEM），10％的过滤青霉素 - 链霉素股票热灭活FBS和1/100稀释。这通常应使用新鲜，但是可以存储在4℃下1个月。 </li><li>每瓶VEGFR酪氨酸激酶抑制剂II（TKIⅡ）在DMSO为原料浓度为5mg / ml，且稀释库存与cDMEM使工作溶液。最后CON中心定位为这项研究是在cDMEM 1.875微克/毫升。根据该公司的建议，储备溶液是稳定长达6个月的，在-20℃下。至于工作的解决方案，使新鲜和在同一天使用。 注意：此药物在工作溶液的浓度应比所需的终浓度2倍高（因为它将稀释两倍以液滴）。与此同时，准备cDMEM含有DMSO中为“控制”处理相同体积相同体积。 </li><li>在蒸馏水中单独准备的尾部消化试剂（50毫米氢氧化钠和1M的Tris-HCl [pH值为8.0）。 </li><li>制作的XY PCR引物25μm的股票（XY正向5&#39;-TGA AGC TTT TGG CTT TGA G-3&#39;和XY逆向5&#39;-CCG CTG C​​CA AAT TCT TTG G-3&#39;）一起在无核酸酶水。 </li><li>准备50X TAE缓冲液（242克Trizma碱57.1冰醋酸，加入100毫升0.5M的EDTA，pH值8.5，加水至1升网络纳尔体积）。制备1X TAE电泳缓冲液（20 ml的50X TAE用水稀释至1L总体积）。 </li><li>要创建2.5％琼脂糖溶液（0.625克琼脂糖加入0.5ml 50X TAE缓冲24.5毫升的水），热琼脂糖溶液在微波炉直至沸腾，然后放凉至约55-65℃（能够触摸）。冷却后，加入2微升1％溴化乙锭溶液，倒入凝胶。 注意！溴化乙锭是一种诱变剂和致畸;请用丁腈手套和处理时应适当的安全协议。备选地，其他潜在毒性较低的凝胶拆解试剂或染料都可以使用。 </li><li>制备封闭液（PBS含有0.1％的Triton X-100，10％胎牛血清[FBS]，和3％牛血清白蛋白[BSA]），用于免疫荧光。 </li><li>制备用于免疫荧光洗液（PBS含有0.1％的Triton X-100，1％FBS，和3％BSA）中。 </li><li>制备PBTx（PBS含有0.1％的Triton X-100），用于免疫荧光。 </li><li>准备孵化室对Culture。补大（直径100mm）的培养皿用35ml高压灭菌的水。广场不远的地方清扫和文化将被执行。 </li></ol>来自小家鼠 2.隔离胎儿睾丸<ol><li>检查雌性小鼠的阴栓每天早晨。当天中午，在其阴道塞检测被认为是E0.5。安乐死的怀孕雌鼠在E11.5，与经批准的动物协议的方式。 </li><li>喷向下小鼠腹部用70％乙醇。 </li><li>打开腹部的皮肤和腹膜使用细剪刀V形切口拉回组织瓣，露出内部器官。 </li><li>定位在卵巢子宫角的两端。用剪刀切下卵巢和结缔组织分离含有从母体胚胎的子宫。 </li><li>轻轻切割胎盘相对的一侧，露出了卵黄囊打开子宫壁。要小心，不要PUncture卵黄囊，以避免损坏或丢失的胚胎。 </li><li>附近的胎盘切去除含胚胎卵黄囊，将它们放入含有PBS与钙和镁的一道菜。钙和镁离子的存在将有助于支持细胞粘附分子来维持组织架构和防止组织粘到清扫工具。 </li><li>用钳子，通过仔细穿刺卵黄囊创建其中胚可以滑出空穴除去卵黄囊和羊膜从胚胎。一旦胚胎是卵黄囊之外，剪断脐带。 </li><li>从现在开始，使用位于无菌组织培养罩显微镜。除去胚的头部，并通过与颈部的两侧镊子夹持丢弃。 </li><li>取出尾部并放入新的离心管XY PCR分析，以确定胚胎的性别。 注意：虽然它是最优的培养性腺尽快收获后，它也是POSsible去除尾部DNA和前培养执行XX / XY基因分型，以使每个胚的性别是已知的，只有所需的性需要培养。而基因型正在做，胚胎可以保持分开（以使它们能够被追踪）在4℃或在冰上直至准备培养。一个需要注意的是，这期间以外在子宫内或培养条件下培养过程中可能潜在地影响生存能力培养或后续开发。 </li><li>钉扎胎的腋下有一对钳（通常在弱手持镊子）固定在对培养皿底部的仰卧位的胚胎。保持这些钳到位，在整个解剖过程中仍然保持胚胎。 </li><li>与其他对镊子，去除皮肤覆盖腹部，轻轻取出肝，肠，和其他器官暴露背部体壁。 </li><li>作为性腺将位于背面壁体上的背主动脉的任一侧，沿着主体的中线运行的大血管，舀泌尿生殖脊封闭镊子下方并通过打开钳子和抬起除去泌尿生殖脊。由于性腺将被松散地贴在墙壁上，注意不要拉涨过快不删除这些连接或性腺可能会拉长，造成损害的器官。 </li><li>移动泌尿生殖嵴为cDMEM在培养皿中，以适应组织的媒体。 </li><li>使用27G的针头泌尿生殖嵴的其余部分分隔开性腺 - 中肾复杂。使用一个针向下压切，并使用其他正确引导所述组织，以允许最佳分离。避免使用对盘底部的锯切运动，锐利的针头会造成塑料碎片，能坚持到组织。请一定要保持完全附着于性腺中肾。 </li></ol> 3.培养性腺的具有小分子抑制剂<ol><li>将两个20μ升移液器每次15微升。切成约1-2毫米断使用干净，灭菌刀片为性腺和加法的控制cDMEM转移阻挡枪头之一，而另一个吸移管将仅用于药物处理cDMEM。 </li><li>排队性腺与他们平行的枪头长轴这样他们就可以很容易地使用截止尖吸管。小心性腺粘附在移液管尖的内部。 </li><li>标号一侧作为控制液滴和其它如含药微滴。只有两个液滴可容纳在35毫米盘的盖子。确保盖子上的标签匹配含尾组织的离心管中的标签。 </li><li>吸移管将15μlcDMEM的含有单个性腺成在小（35毫米）培养皿的盖的液滴（只使用盖，由于底部是太高，以适应加湿培养皿腔内）。将两个独立的含性腺液滴上的菜的两侧，以及分隔FROM彼此。检查显微镜下，以确保性腺已转移到液滴。 </li><li>到指定为控制所述液滴，增加额外的15微升cDMEM含有DMSO（在步骤1.5制），使用30微升总体积的液滴。仅使用“控制”吸管，不会接触毒品。 </li><li>的其他液滴（药物处理的样品）中，添加15微升含TKI-ⅡcDMEM，使用30微升总体积的液滴。只能使用指定药物处理的样品的吸管。 </li><li>使用吸管，摊开液滴在不断扩大的圆形图案，直到他们约15-18毫米，直径性腺位于大致在中间。定向所述性腺使得其位于在其一侧和性腺和中肾很容易分辨。定向所述肺，使得两个裂片平放。 <ol><li>虽然液滴直径可能会略有不同，确保性腺都没有莲花灯克和在适当位置由表面张力保持。确保液滴不相互接触或盖的一侧。如果没有表面张力，该机构将在培养过程中生长到盖和扁平化，从而扭曲器官的形态。 </li></ol></li><li>小心地将含微滴培养皿盖直立到水在大（100毫米）加湿器皿的表面上。确保没有气泡截留在盖的底部下方，并且它平放的水的表面上。不要反转盖子，注意不要让水接触到盖洗脱液滴位于顶部。 </li><li>要创建一个小的，潮湿的室内，应立即用较大的加湿器盘的盖中盖围住性腺文化。法尽快尽量减少任何媒体蒸发。确保更小的菜都有它和较大盘的盖子之间的空间自由走动;否则，冷凝可能使密封和b锁空气交换的组织。 </li><li>一旦两个小盖子被放置在室中，紧接在室放入孵化器。孵育液滴培养48小时，在湿润的 CO 2培养箱中在37℃。 </li></ol> 4.聚合酶链反应确定胚胎的性别<ol><li>添加胚胎尾部到1.5ml微量离心管的底部。 </li><li>添加到每个管200微升的50mM NaOH中。 </li><li>放置在95℃下15分钟，或直至组织完全溶解。 </li><li>加入50微升1M的Tris-HCl和旋涡轻轻简单。 </li><li>加入0.5μl25μM的引物溶液，2.5微升10X Taq缓冲液，0.5微升的10mM的dNTP（核苷酸），19.3微升核酸酶：在1.5ml微量离心管，通过每体积由样本的总数乘以一个最新的PCR主混合物免费的水，0.2微升DNA Taq聚合酶。拌匀。 </li><li>添加23微升PCR主混合物以PCR含有消化尾3微升裂解液管。 </li><li>轻弹管将它们混合，然后执行短的自旋使样品管的底部。 </li><li>运行XY（短）PCR程序（ 表1）。 </li><li>加载样品（与5倍的染料）和梯在1X TAE缓冲液在2.5％琼脂糖凝胶。 </li><li>运行琼脂糖凝胶电泳直到XX和XY波段可以解决。 </li><li>图像的紫外线和捕获图像的凝胶。 </li><li>分析的X染色体特异性VS Y染色体特异条带（X染色体= 331个碱基对[BP]和XY = 302基点）13; XY（男性）的样品将有331两个频段和302基点，而XX（女性）的样本将显示为一个单一的331 bp的条带。 </li></ol> 5.整装器官免疫第1天： <ol><li>使用1000微升吸管截止尖取下文化性腺。如果需要的话，它们可以是放置在PBS一碟洗掉培养基和试剂。放入PBS中于0.5 ml离心管。较小的管子尺寸将节约试剂和更容易地跟踪样本在管。 <ol><li>一定要保持控制和处理的样品，以及XX和XY样品，在不同的试管和文化与移液器单独设置删除它们，以避免潜在的药物污染。 </li></ol></li><li>用PBS洗两次性腺。从这点上，该协议可以使用1×PBS缺乏钙和镁。把它们洗干净，允许性腺沉到管（通过重力）的底部，然后除去上面的液体。小心不要吸取太靠近他们失去性腺。 </li><li>从管中除去尽可能多的PBS作为可能的。加入250微升4％多聚甲醛的PBS中含有0.1％的Triton X-100，和让孵育O / N在4℃下于一个摇杆。或者，该固定可以在4℃上的摇杆进行2小时。注意！ Paraformaldehyde是一种有毒物质，因此在处理时应遵守安全协议。 </li></ol>第2天： <ol start="4"><li>在250微升PBTx在室温两次冲洗性腺。 </li><li>用250μlPBTx，每次10分钟，在室温上的摇杆洗性腺3次。 </li><li>方框性腺至少1小时在250微升阻断在RT上的摇杆溶液。 </li><li>染色性腺O / N，在4℃在250微升的第一抗体稀释在封闭液摇臂。 </li></ol>第3天： <ol start="8"><li>在250微升洗液冲洗两次性腺。 </li><li>洗性腺3次用250μl洗涤液用于在每个摇杆10分钟，在室温。 </li><li>块中的性腺1小时的250微升阻断在RT上的摇杆解决方案。 </li><li>盖上离心管铝箔保护荧光的二抗避光。 </li><li>孵育性腺2-4小时，在RT上ARocker在稀释于封闭含赫斯特33342或者解决方案250微升二抗，摇臂上进行二次抗体和赫斯特33342孵化O / N在4℃。 </li><li>在250微升PBTx两次冲洗性腺。 </li><li>在250微升PBTx，每次10分钟，在室温上的摇杆洗性腺3次。 </li><li>使用截止的枪头，转移性腺到最小的液体幻灯片。去除多余的液体，吸液管，但是要确保组织不会干。 </li><li>东方在用钳子希望的方式性腺，赶紧加入一滴安装介质安装幻灯片上的性腺。确保安装介质大衣完全所有的样品;关键是要避免让样品干燥。 </li><li>适当大小使用盖玻片（1.5号方式），轻轻盖样品。让安装介质传播来联系盖玻片的整个表面上。如果有必要，非常轻按盖玻片，这样的角落有没有大的气泡。 </li><li>密封与周围的边缘清晰指甲油幻灯片降低安装介质的蒸发。商店安装载玻片在黑暗中在4℃。 </li></ol>

<ol>
	<li>Kalaskar, V. K., Lauderdale, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+development+in+a+serum-free+whole+embryo+culture+system.">Mouse embryonic development in a serum-free whole embryo culture system.</a> J. Vis. Exp. (85), e50803 (2014).</li><li>Maatouk, 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=Stabilization+of+beta-catenin+in+XY+gonads+causes+male-to-female+sex-reversal.">Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal.</a> Hum. Mol. Genet. 17, 2949-2955 (2008).</li><li>DeFalco, T., Bhattacharya, I., Williams, A. V., Sams, D. M., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yolk-sac-derived+macrophages+regulate+fetal+testis+vascularization+and+morphogenesis.">Yolk-sac-derived macrophages regulate fetal testis vascularization and morphogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 111, E2384-E2393 (2014).</li><li>Coveney, D., Cool, J., Oliver, T., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+analysis+of+vascularization+during+primary+development+of+an+organ,+the+gonad.">Four-dimensional analysis of vascularization during primary development of an organ, the gonad.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7212-7217 (2008).</li><li>Walton, K. D., Kolterud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fetal+whole+intestine+culture+system+for+ex+vivo+manipulation+of+signaling+pathways+and+three-dimensional+live+imaging+of+villus+development.">Mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development.</a> J. Vis. Exp. (91), e51817 (2014).</li><li>Delmarcelle, A. S., Villacorte, M., Hick, A. C., Pierreux, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+culture+system+to+study+thyroid+development.">An ex vivo culture system to study thyroid development.</a> J. Vis. Exp. (88), e51641 (2014).</li><li>Costantini, F., Watanabe, T., Lu, B., Chi, X., Srinivas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+embryonic+mouse+kidney,+culture+in+vitro,+and+imaging+of+the+developing+organ.">Dissection of embryonic mouse kidney, culture in vitro, and imaging of the developing organ.</a> Cold Spring Harb. Protoc. 2011, (2011).</li><li>Petzold, K. M., Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+ex+vivo+culturing+of+embryonic+pancreas.">A system for ex vivo culturing of embryonic pancreas.</a> J. Vis. Exp. (66), e3979 (2012).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J. Vis. Exp. (51), e2720 (2011).</li><li>Combes, A. N., 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=et+al.Endothelial+cell+migration+directs+testis+cord+formation.">et al.Endothelial cell migration directs testis cord formation.</a> Dev. Biol. 326, 112-120 (2009).</li><li>Bott, R. C., McFee, R. M., Clopton, D. T., Toombs, C., Cupp, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+and+kinase+domain+region+receptor+are+involved+in+both+seminiferous+cord+formation+and+vascular+development+during+testis+morphogenesis+in+the+rat.">Vascular endothelial growth factor and kinase domain region receptor are involved in both seminiferous cord formation and vascular development during testis morphogenesis in the rat.</a> Biol. Reprod. 75, 56-67 (2006).</li><li>Cool, J., DeFalco, T. J., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular-mesenchymal+cross-talk+through+Vegf+and+Pdgf+drives+organ+patterning.">Vascular-mesenchymal cross-talk through Vegf and Pdgf drives organ patterning.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 167-172 (2011).</li><li>Clapcote, S. J., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplex+PCR+assay+for+sex+determination+in+mice.">Simplex PCR assay for sex determination in mice.</a> BioTechniques. 38, 702, 704-706 (2005).</li><li>Nel-Themaat, 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=et+al.Morphometric+Analysis+of+Testis+Cord+Formation+in.">et al.Morphometric Analysis of Testis Cord Formation in.</a> Dev. Dyn. 238, 1100-1110 (2009).</li><li>Kent, J., Wheatley, S. C., Andrews, J. E., Sinclair, A. H., Koopman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+male-specific+role+for+SOX9+in+vertebrate+sex+determination.">A male-specific role for SOX9 in vertebrate sex determination.</a> Development. 122, 2813-2822 (1996).</li><li>Morais da Silva, 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=et+al.Sox9+expression+during+gonadal+development+implies+a+conserved+role+for+the+gene+in+testis+differentiation+in+mammals+and+birds.">et al.Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds.</a> 14, 62-68 (1996).</li><li>Rockich, B. 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=Sox9+plays+multiple+roles+in+the+lung+epithelium+during+branching+morphogenesis.">Sox9 plays multiple roles in the lung epithelium during branching morphogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 110, E4456-E4464 (2013).</li><li>Si-Tayeb, K., Lemaigre, F. P., Duncan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+and+development+of+the+liver.">Organogenesis and development of the liver.</a> Dev. Cell. 18, 175-189 (2010).</li><li>Brennan, J., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+tissue,+two+fates:+Molecular+genetic+events+that+underlie+testis+versus+ovary+development.">One tissue, two fates: Molecular genetic events that underlie testis versus ovary development.</a> Nat. Rev. Genet. 5, 509-521 (2004).</li><li>Martineau, J., Nordqvist, K., Tilmann, C., Lovell-Badge, R., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-specific+cell+migration+into+the+developing+gonad.">Male-specific cell migration into the developing gonad.</a> Curr. Biol. 7, 958-968 (1997).</li><li>Yokonishi, T., Sato, T., Katagiri, K., Ogawa, 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+vitro+spermatogenesis+using+an+organ+culture+technique.">In vitro spermatogenesis using an organ culture technique.</a> Methods Mol. Biol. 927, 479-488 (2013).</li><li>Sato, T., 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=In+vitro+production+of+functional+sperm+in+cultured+neonatal+mouse+testes.">In vitro production of functional sperm in cultured neonatal mouse testes.</a> Nature. 471, 504-507 (2011).</li><li>Gohbara, 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=In+vitro+murine+spermatogenesis+in+an+organ+culture+system.">In vitro murine spermatogenesis in an organ culture system.</a> Biol. Reprod. 83, 261-267 (2010).</li></ol>

The authors declare that they have no competing financial interests.

这项研究表明，具有研究胎儿发育许多潜在的应用离体全器官液滴的方法。这种技术可以用于多种器官，和允许研究者解决生物学问题，是很难使用体内检查方法由于胚胎和潜在胚胎致死的交通不便。此培养方法比其他体外附加利益的方法，如哺乳动物细胞系：整个器官都可以使用，因此维持存在于子宫内临界细胞间的相互作用;而培养体积很小（约30微升），因?...

在体内在人类器官再生是非常有限的;因此，组织工程，组织和从由主机捐献单个细胞器官的发育，是变器官更换一个有吸引力的潜在疗法。但是，对于该治疗策略是成功的，因素和参与器官的形态形成的细胞相互作用，必须彻底的研究和易于理解的。由于无法研究特定器官与传统方法的发展，研究人员已转向替代全胚胎或整个器官培养。 Kalaskar 等人 1已经表明， 体外全胚胎培养得到类似的结果（在培养的胚胎的58％），以在子宫内发育，表明体外培养方法可用于器官发生研究一个可行的替代方案。 个性化的器官培养系统，例如本体外的dro诗人盖伊培养系统，允许对整个器官分析独立的全身效应，而通过加药理学试剂或抗体的允许操作的特定信号传导途径或细胞的相互作用。传统上，胎儿器官发育的研究已不限于转基因和基因敲除小鼠的技术中，除了递送母系药理试剂。但是，也有涉及这些技术和治疗方法在体内的技术问题;最关注围绕影响各种器官同时经常导致胚胎致死性的影响。研究操纵胎儿发育的另外一个关注的药理是药物对子宫内胚胎发育的母体效应（如药物产妇代谢之前到达胚胎），如果这样的试剂可以穿过胎盘屏障。 整个器官培养技术说明这里被改编自协议首先通过Maatouk 等人 2描述，其中整个胎儿性腺孵育在体外直立液滴培养物。培养的胎儿性腺之一显著优点是，小分子抑制剂能够通过简单的扩散容易地访问整个器官。 。DeFalco等人已经表明，利用在与小分子抑制剂结合本体外液滴培养方法可用于研究信令进程和性腺发育3期间发生相互作用;这些过程将难以检查体内由于技术上的挑战（例如，药物通过胎盘或影响使用基因或药理学方法多个器官杀伤力通路）。 液滴文化不仅在某些方面比在子宫内的实验改进，而且它是一个改进， 在体外和前六VO的系统。因为它们缺乏多样的细胞类型，缺乏临界外基质（ECM）组件，其允许器官结构的形成，并且可以在信号级联放大显示的工件的使用细胞系，研究形态发生是极为困难的。虽然组织工程在创造支架模拟ECM做显著的改善，对于缺乏知识的哪些信号需要通过器官中的细胞类型使得它具有挑战性的在体外建立一个器官系统。其它体外系统已被预先建立，研究器官，或更具体形态发生，并已为胎儿器官的琼脂4实时成像非常成功的，转孔5，过滤器6，和其他支架矩阵7,8。液滴培养系统的优点是，它允许形态的研究通过提供利用较少的试剂，其分别为邻的能力FTEN昂贵，而且还给予器官表面张力，这是生长和信令能力9重要。 在小鼠中，初始睾丸形态取胚胎（E）之间发生级E11.5和E13.5;这些阶段包括的最佳时间窗口用于检查影响性别特异性分化因子。间期间睾丸形成发生的关键过程是睾丸帘线结构的产生和睾丸特异性的血管网络的形成。利用这种体外全器官液滴培养系统，一个是能够改变雄性特异性血管化和通过使用小分子抑制剂的抑制睾丸形态发生阻断了受体的血管内皮生长因子（VEGF）的活性;血管内皮生长因子介导的血管重构是睾丸发育10-12的关键。这种技术可以成功地应用于其它器官，并且可以针对特定的时间发展的窗口。全安装器官成像允许重要结构的可视化以及来自各种抑制剂的给药引起的结构和细胞变化。重要的是，这个系统是在研究人员可以绕过从母体药品监督管理部门或系统中断期间体内靶基因战略的潜在混杂影响是有利的。因此，这整个器官离体液滴培养系统可以显著改善了解哪些胎儿发育过程中特别会出现特定的器官内的相互作用和信号的能力。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses: Squares (22 mm x 22 mm, No. 1.5)</td>
			<td>Fisherbrand</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sally Hansen Xtreme Wear Nail Polish, Invisible</td>
			<td>Sally Hansen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Strip 0.2 mL PCR Tubes &#38; Detached Flat Caps</td>
			<td>GeneMate</td>
			<td>T3218-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L P1000L, P200L, P20L, P10L, P2L</td>
			<td>Gilson</td>
			<td>FA10006M, FA10005M, FA10003M, FA10002M, FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 ml microcentrifuge tubes</td>
			<td>Denville</td>
			<td>C2170</td>
			<td></td>
		</tr>
		<tr>
			<td>Posi-Click 0.6 ml microcentrifuge tubes</td>
			<td>Denville</td>
			<td>C2176</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1096FR</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1122</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe with 27gauge needles</td>
			<td>BD PrecisionGlide</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305559</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#956;M PES syringe filter</td>
			<td>VWR</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Grade 3 Qualitative Filter Paper Standard Grade, circle, 185 mm</td>
			<td>Whatman</td>
			<td>1003-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 35mm Easy Grip Style Cell Culture Dish</td>
			<td>Falcon/Corning</td>
			<td>353801</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes, Sterile (100 mm x 15 mm)</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Galaxy 14 S CO2 Incubator</td>
			<td>Eppendorf</td>
			<td>CO14S-120-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>Nuare</td>
			<td>NU-425-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-centrifuge&#160;</td>
			<td>Fisher Scientific</td>
			<td>05-090-100</td>
			<td></td>
		</tr>
		<tr>
			<td>BioExpress GyroMixer Variable XL</td>
			<td>GeneMate</td>
			<td>R-3200-1XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler Pro Thermal Cycler with control panel</td>
			<td>Eppendorf</td>
			<td>950040015</td>
			<td></td>
		</tr>
		<tr>
			<td>SMZ445 stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ445</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiImage Light Cabinet with AlphaEase Software</td>
			<td>Alpha Innotech Corporation</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute 200 proof Ethanol</td>
			<td>Fisher</td>
			<td>BP2818-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Dibasic MW 142) Na2HPO4</td>
			<td>Fisher</td>
			<td>S374-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (Monobasic MW 136) KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M2393-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Sigma</td>
			<td>C5670-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion Nuclease-Free Water</td>
			<td>Life Technologies</td>
			<td>AM9938&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>XY PCR Primer&#160;</td>
			<td>IDT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher</td>
			<td>A38-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine Tetraacetic Acid (EDTA)</td>
			<td>Fisher</td>
			<td>BP2482-1</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Ethidium bromide solution</td>
			<td>Fisher</td>
			<td>BP1302-10</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>GeneMate</td>
			<td>E-3120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>367176-2.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set, 100 mM Solutions</td>
			<td>Thermo Scientific</td>
			<td>R0182</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Choice Taq polymerase with 10x Buffer</td>
			<td>Denville</td>
			<td>CB-4050-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>FluorMount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Chloride (HCl)</td>
			<td>Fisher</td>
			<td>A144212</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), powder, Fraction V, Heat shock isolation</td>
			<td>Bioexpress</td>
			<td>0332-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), triple 100-nm filtered</td>
			<td>Fisher</td>
			<td>03-600-511</td>
			<td>Heat-inactivate before using</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Use at 1:100</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), Hybri-max, sterile-filtered</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGFR Tyrosine Kinase Inhibitor II - CAS 269390-69-4 - Calbiochem</td>
			<td>EMD Millipore</td>
			<td>676481</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Sox9 Antibody</td>
			<td>Millipore</td>
			<td>AB5535</td>
			<td>Use at dilution: 1:4,000</td>
		</tr>
		<tr>
			<td>Rat Anti-Mouse PECAM1 (CD31) Antibody</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>Use at dilution: 1:250</td>
		</tr>
		<tr>
			<td>Rabbit Cleaved Caspase-3 (Asp175) Antibody</td>
			<td>Cell Signaling</td>
			<td>9661S</td>
			<td>Use at dilution: 1:250</td>
		</tr>
		<tr>
			<td>Rat E-cadherin / CDH1 Antibody (ECCD-2)</td>
			<td>Life Technologies</td>
			<td>13-1900</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Hoechst 3342, trihydrochloride, trihydrate</td>
			<td>Invitrogen (Molecular Probes)</td>
			<td>H1399</td>
			<td>Use at 2ug/ml</td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Rat IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>712-165-153</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>712-605-153</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>Life Technologies</td>
			<td>A31572</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A21206</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A21208</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td>Use at dilution: 1:500</td>
		</tr>
	</tbody>
</table>

using ex vivo upright droplet cultures of whole fetal organs to study developmental processes during mouse organogenesis

在子宫内调查器官是胎盘哺乳动物，由于试剂的交通不便在技术上具有挑战性的过程中对子宫内的发育胚胎。新开发的体外直立滴培养法提供了一个有吸引力的替代在子宫内进行的研究。 体外液滴文化提供了检查和处理，通过使用各种保护和活化化合物的细胞相互作用和多样化的信号通路的能力;另外地，可以研究在特定器官的发展多种药理试剂的作用，而不在子宫内的全身药物递送的不需要的副作用。相对于其他的体外系统中，微滴培养不仅允许研究三维形态发生和细胞-细胞相互作用的能力，这是不能在哺乳动物细胞系被再现，同时还要求显著少řeagents比其他离体和体外协议。本文示范了正确的小鼠胎儿器官解剖和直立液滴培养技术，其次是全器官免疫证明该方法的有效性。 体外液滴培养方法允许形成器官结构可比所观察到的体内 ，并且可以用来研究由于胚胎致死性在体内模型中 ，否则难以研究的过程。作为一个模型应用系统中，小分子抑制剂将用于探测血管的睾丸形态发生中的作用。此体外液滴培养方法是可扩展到其他胎儿器官系统，如肺和潜在别人，虽然每个器官必须是广泛的研究，以确定任何器官特异性修改的协议。这个器官培养系统提供了在实验与胎儿各器官的灵活性，并导致obtained。使用这种技术将有助于研究人员深入了解胎儿的发育。

体外培养液滴允许一个操纵整个器官，如生殖腺，来研究细胞相互作用和动力学。 图1展示了在一个逐步的方式如何准备E11.5性腺液滴培养。在培养协议的第一个步骤包括最初除去从母小鼠（图1A和1B）的含胚子宫。除去从母亲子宫后，子宫壁被切断，将胚胎从卵黄囊到PBS中用于进一步解剖（图1C-E）的解放。去除内脏器官的后?...

Watch this Scientific Journal Video about Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis at JoVE.com

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

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Research

JoVE Journal

Developmental Biology

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

12.4K 次观看.  Cincinnati Children's Hospital Medical Center. The ex vivo upright droplet culture is an alternative to current in vitro and in vivo experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

视频: Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

科研

发育生物学

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

使用主神经球文化研究纤毛

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

拟南芥花器官和角果的全装清和染色

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

人体外伤模型和全山染色方法，以准确评估皮肤修复

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

从小鼠面部过程和培养的腭间充质细胞中分离全细胞蛋白质裂解物以进行磷酸化蛋白分析

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...