我们感谢贾格布博士、j. Salvi 博士和 Petelski 为本议定书所依据的原始研究所作的贡献。我们还感谢美洲骆驼和 w Makmura 提供技术援助和畜牧业。我们承认 NIDCD 培训补助金 T32 DC009975, NIDCD 赠款 R01DC015530, 罗伯逊治疗发展基金, 和家庭基金会的资金。最后, 我们承认霍华德休斯医学研究所的支持, Hudspeth 博士是一名调查员。

这里描述的所有方法都得到了洛克菲勒大学和南加州大学动物保育和使用委员会的批准。
1. (可选) 用小鼠尾肌腱制备胶原 i 型溶液
注意:胶原蛋白 I 解决方案可在商业上使用。按照制造商的说明进行凝胶制剂。
<ol><li>弄死 5-10 个年轻成人 (3-5 周大) 的小鼠, 任何野生型菌株与二氧化碳按照有关机构动物护理和使用委员会批准的议定书16。收集尾巴和消毒他们通过淹没在70% 乙醇至少在室温下4小时。 注意:培养超过48小时应避免, 因为它导致过多的组织脱水和阻碍肌腱提取过程。</li>
	<li>从每条尾巴的皮肤, 通过引入一个切割刀刀片和缩回整个皮肤的镊子。将剥皮的尾巴转移到一个100毫米的培养皿中, 里面装满干净的70% 乙醇, 切成10毫米的段。 注意:丢弃尾部最薄的部分, 这是难以操作的。</li>
	<li>使用镊子, 确保尾部的每一段到培养皿的底部, 并使用第二对精钳从尾部一次提取肌腱。个别肌腱纤维应出现的阻力最小。 注意:陈旧的, 沉闷的 #5 钳为这一步的工作很好。</li>
	<li>在不育的分子级水中, 准备100毫升的醋酸0.1% 溶液 (按体积), 并在不育的100毫米培养皿中加入10毫升的溶液。将肌腱转移到培养皿中, 在室温下留1小时变性胶原蛋白 i。</li>
	<li>使用无菌手术刀或虹膜剪刀钝, 弯曲的提示, 以切碎肌腱成 1-2 毫米碎片。将切碎的肌腱转移到不育的50毫升管中, 加入0.1% 醋酸, 使体积增加到50毫升。 注意:使用四或五尾为每50毫升的酸, 以实现胶原 i 浓度的 2.0-2.5 毫克/毫升。</li>
	<li>将胶原蛋白 i 溶液冷藏4摄氏度, 最小为48小时, 以促进蛋白质的完全变性。涡旋与桌面涡设置为最大速度为1分钟两次一天。</li>
	<li>用二辛可宁酸法测定溶液的蛋白质浓度, 通过添加0.1% 醋酸溶液, 将胶原 i 浓度调整为 2.0-2.5 毫克/毫升, 并贮存在4摄氏度。 注意:胶原 i 溶液可以储存一到两年。</li>
	<li>选离心的胶原 i 溶液在 2000 x g 1 小时在4摄氏度, 并使用半透明的顶部分数 (约一半的体积), 以实现光学明确的胶原凝胶在4节。</li>
</ol>2. 前庭和听觉器官的解剖
<ol><li>根据有关机构动物保育和使用委员会批准的议定书, 弄死怀孕小鼠任何与二氧化碳有关的菌株16。提取并斩首胚胎。 注意:Lfng-CreERT2/tdTomato 鼠标可用于在暴露于 4-hydroxytamoxifen17时允许永久标记支持单元格。</li>
	<li>消毒所有的工作表面和解剖器械, 包括两对 #5 钳和一条发刀18, 通过清洁70% 乙醇。</li>
	<li>通过采用无菌手术刀刀片或使用两对 #5 钳, 将每个头分成两半。提取的颞骨, 其中包含内耳19, 并把它们放入15毫升的冰冷汉克的平衡盐溶液 (HBSS) 在60毫米的培养皿。把盘子放在冰上。 注意:从 E13.5 到 E18.5 的各个阶段的胚胎已经成功使用。</li>
	<li>选在50毫升的锥形管中轻轻摇动它们, 其中包含30毫升冰冷的 HBSS, 并更换 HBSS 三或四倍。把管子放在冰上。 注意:这一步骤限制了可能的组织培养的污染, 并迅速带来的温度为4摄氏度, 这可以防止组织退化和细胞死亡。</li>
	<li>使用两对精细 #5 钳, 分开的内耳从颞骨和转移的耳朵, 每次三或四, 成一个60毫米的培养皿充满冰冷的 HBSS。</li>
	<li>解剖前庭感官器官。<ol>
<li>将耳朵内侧侧向上定位, 并找到囊。用两对 #5 钳, 除去前庭器官周围的软骨。轻轻地切断前庭神经, 囊和囊之间的连接, 以及半圆形运河, 如图 1所示。轻轻拉囊和 ampullae 的上级和水平半圆运河从耳朵。 注意:该方法可用于 E16.5 E18.5 阶段的内耳。保存下面3节的软骨碎片。</li>
	</ol>
</li>
	<li>解剖耳蜗。<ol>
<li>用两对 #5 钳去除听觉器官周围的软骨组织。轻轻地切断耳蜗底座和囊之间的连接, 如图 1所示。 注意:对于分期 E13.5-E14.5, 软化软骨前解剖通过治疗内耳与0.25% 胶原酶 I 在磷酸盐缓冲盐水 (PBS) 溶液5分钟室温。保存下面3节的软骨碎片。</li>
	</ol>
</li>
	<li>使用 200-µL 吸管装有宽的不粘锅尖端, 转移胞果和耳蜗到一个30毫米培养皿填充生长培养基, 包括 DMEM/F12 补充33毫米 Dglucose, 19 毫米碳酸氢钠, 15 毫米 4 (2 羟乙基)-1piperazineethanesulfonic 酸 (HEPES), 1 毫米谷氨酰胺, 1 毫米烟酰胺, 20 毫克/升表皮生长因子, 20 毫克/升成纤维细胞生长因子, 10 毫克/升胰岛素, 5.5 毫克/升转铁蛋白, 5 µg/升亚硒钠。</li>
	<li>保持囊的准备为3小时, 在37摄氏度的组织培养孵化器与5% 二氧化碳, 以允许愈合的切口在解剖过程中引入的上皮。耳蜗的准备工作应转移到胶原凝胶后10分钟的解剖。 注意:因为解剖是在冰冷的 HBSS 中进行的, 所以没有必要将生长培养基预热到37摄氏度。</li>
</ol>3. (可选) 通过增加不同浓度的软骨细胞来调节胶原蛋白 i 凝胶的硬度
注意:软骨细胞隔离的方法从戈塞特et 。20
<ol><li>准备1% 溶液的胶原酶 I 在无菌 1x PBS 和存储 100-200 µL 整除数在-80 摄氏度。在冰上解冻时需要, 避免多个冻融循环。</li>
	<li>用细钳收集从前庭和听觉器官解剖遗留下来的软骨碎片, 从 10-12 耳。从软骨中分离结缔组织和内耳组织。将软骨转移到30毫米培养皿中, 加入300µL 的无菌生长培养基。</li>
	<li>使用无菌手术刀刀片或虹膜剪刀钝弯曲提示, 以粉碎组织, 以达到大约0.5 毫米的软骨片长度。</li>
	<li>添加胶原酶 I 到培养基与软骨, 以达到最终的酶浓度为0.25%。把盘子转移到一个组织培养孵化器在37°c 毒气与5% 二氧化碳。吸管积极使用1000µL 吸管每20分钟, 直到软骨部分离解, 并已不再区分在解决方案, 然后孵化额外20分钟。 注意:在囊准备的情况下, 可以在步骤2.9 中执行软骨细胞隔离;它需要大约2小时 (3-4 吹打回合)。</li>
	<li>收集细胞悬浮在一个15毫升圆锥管和调整体积为10毫升与无菌 1x PBS。离心机在 800 x g 5 分钟在4摄氏度。取出上清液, 并用重悬10毫升无菌 1x PBS 中的细胞。离心机在 800 x g 5 分钟在4°c 和去除上清。 注意:两次冲洗细胞是至关重要的;任何剩余的胶原酶, 我会消化胶原蛋白 i 凝胶。</li>
	<li>使用 200-µL 吸管, 添加100µL 培养基的细胞颗粒和吸管轻轻地并用重悬。使用 hemocytometer 计算单元格并在冰上维持它们。</li>
	<li>为了增加凝胶的硬度, 添加软骨细胞到中性胶原 i 凝胶溶液 (1 节), 并迅速混合在整个凝胶之前分配细胞凝固。 注意:胶原 i 型凝胶的弹性模量 (硬度的测量), 随着软骨细胞数量的增加而增大11。该关系由实验确定的线性函数E = 206·描述。Nc + 15, 其中E是帕斯卡中的弹性模数, Nc是数以百万计的软骨细胞数。有关凝胶刚度测量的详细协议, 请参阅原始文章11。</li>
</ol>4. 将前庭或听觉感觉器官放置在胶原蛋白 i 凝胶中
<ol><li>在不育的1.5 毫升管, 制备胶原 i 聚合溶液的混合160µL 10x PBS 与苯酚红色 pH 值, 133 µL 0.34 米氢氧化钠, 70 µL 的0.9 米碳酸氢钠, 40 µL 1 米 HEPES。把管子放在冰上。 注意:这个食谱提供了聚合解决方案, 以准备2毫升胶原蛋白 i 凝胶, 但可以根据需要缩放。</li>
	<li>混合100µL 的聚合溶液和400µL 的胶原 i 溶液在冰上轻轻吹打上下在一个冷冻1.5 毫升管。为了增加凝胶的硬度, 添加50µL 的生长培养基中含有软骨细胞, 并轻轻混合, 如第3节所述。</li>
	<li>将中性胶原 i 溶液的500µL 转移到一个冷冻的30毫米培养皿中, 用10毫米的玻璃底部插入物或一个四井板的井。 注意:保持冰上的所有试剂, 防止胶原蛋白的快速和不均匀聚合是至关重要的。</li>
	<li>快速将耳蜗或胞果转移到中性胶原 i 溶液中, 用不育的发刀18或一对精钳将器官调整到所需的位置. 注意:胶原 i 聚合在 1-2 分钟以后变得引人注目, 因为溶液变得混浊。</li>
	<li>在溶液周围的组织已聚合, 放置培养皿或四井板20分钟37°c 在一个组织培养孵化器与5% 二氧化碳毒气, 以确保完全聚合。</li>
	<li>添加3毫升的生长培养基辅以0.5% 胎牛血清 (血清) 每培养皿或500µL 的同一培养基每井四井板。保持37°c 的文化在一个组织培养孵化器与5% 二氧化碳毒气。如果需要, 补充生长培养基与10µM 5-乙炔-2´脱氧尿苷 (教育局) 标签增殖细胞。 注意:如果需要, 可以使用更高的血清浓度。</li>
</ol>5. 三维的前庭和听觉知觉器官的病毒注射
<ol><li>在冰上解冻所需的病毒, 并在0.5 毫升锥形管中与台盼蓝溶液混合, 达到最终染料浓度的0.05%。使用 10-20x 染料, 以避免大量稀释的病毒。保持冰块。 注意:腺病毒5型对感染囊19中的支持细胞最有效, 而腺相关病毒 Anc80 可以感染囊和耳蜗21中的毛发细胞和支持细胞。</li>
	<li>在一双双目解剖显微镜的最高放大倍数下, 用干净的细钳在微拉拔器上折断玻璃针的尖端。 注意:优化拔针器的设置非常重要。针与 9-12 毫米小腿和 20-30-µm 开口工作最好。</li>
	<li>从孵化器中移除感官器官的培养。将针贴在 microinjector 上, 用 2-3 µL 染料和病毒混合物填充。在双目解剖显微镜下观察, 将针头推进感官器官。</li>
	<li>通过三维 utricular 培养的屋顶的间质和上皮层轻轻地驱动针尖。注入病毒混合物, 直到囊和 ampullae 的空洞填充蓝色染料。 注意:因为它允许容易接触感官器官, 一个10毫米的玻璃底部培养皿是最佳的注射。</li>
	<li>在5% 二氧化碳的组织培养孵化器中孵育37摄氏度。 注意:在病毒结构中使用绿色或红色荧光蛋白时, 荧光在病毒传导后明显24小时。</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension:+using+collective+cell+migration+and+cell+intercalation+to+shape+embryos.">Convergent extension: using collective cell migration and cell intercalation to shape embryos.</a> Dev Camb Engl. 139 (21), 3897-3904 (2012).</li></ol>

作者没有什么可透露的。

研究了在发育过程中调节内耳生长和分化的分子信号5,6,7,8,9,10。然而, 从 utricular 模型系统获得的证据表明, 通过细胞连接感知的机械线索和河马信号的活化, 在这些过程中也扮演着重要的角色2,11,<...

体外系统促进了哺乳动物器官发育的大多数方面的研究。现在有两种主要的方法用于前庭感觉器官的培养: 自由浮动的1和黏附的2准备。这两种方法都允许调查毛细胞漏洞3和再生1,4 在体外。此外, 凹槽5、6、Wnt7、8和表皮生长因子受体 (EGFR)9、10信号级联在内耳中的发展角色有在某种程度上是通过使用体外的感官上皮培养而建立的。然而, 细胞生长和分化的控制, 不仅通过格局来说信号, 而且还通过物理和机械的线索, 如细胞间接触, 僵硬的细胞外基质, 和机械伸展或收缩。这种机械刺激的作用在开发内耳体内的研究中具有挑战性。此外, 现有的游离漂浮和贴附培养方法不适用于这种研究在体外。在这里, 我们描述了一个三维脊髓培养的方法, 胶原蛋白 i 凝胶的变化刚度。此方法主要保留前庭和耳蜗感官器官的体内结构, 并允许对机械力对生长和分化的影响进行调查11。
因为已知的机械刺激可以激活下游分子事件, 如河马信号通路12,13,14,15, 这是很重要的, 能够结合机械刺激生化和基因操控。这里描述的文化方法允许病毒介导的基因交付, 因此可以用来研究在内耳发育过程中的机械和分子信号学11。

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three-dimensional organotypic cultures of vestibular and auditory sensory organs

内耳的感官器官由于无法进行实验操作和光学观察, 对哺乳动物的研究具有挑战性。此外, 虽然现有的文化技术允许生物化学的扰动, 这些方法并没有提供一个方法来研究机械力和组织刚度的影响, 在发展的内耳感官器官。在这里, 我们描述了一个三维脊髓文化的完整的小鼠囊和耳蜗克服这些限制的方法。在这里描述的三维矩阵刚度的调整技术允许操纵的弹性力反对组织生长。因此, 该方法可以用来研究机械力在内耳发育过程中的作用。此外, 这些文化还允许病毒介导的基因传递, 可用于增益和功能损失的实验。这种培养方法保留了固有的毛发细胞和支持细胞, 作为传统的前庭和听觉知觉器官的二维文化的潜在优越替代品。

从胚胎耳中培养的前庭和听觉知觉器官 40-Pa 胶原蛋白 i 型凝胶模拟低刚度胚胎条件11, 保留相对正常的三维结构 (图 1) 并保持毛发细胞和支持单元格 (图 2和图 3)。虽然支持的细胞密度降低了30% 以上 (学生的t测试: n = 4, p < 0.004) 和毛细胞密度下降 60% (学生的t测试: n = 5, ...

Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

三维脊髓培养的小鼠囊和耳蜗在光学透明胶原 i 凝胶保存固有的组织形态学, 允许机械刺激通过调整基质刚度, 并允许病毒介导的基因传递。

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. ...

three-dimensional-organotypic-cultures-vestibular-auditory-sensory

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  Keck School of Medicine of the University of Southern California. 三维脊髓培养的小鼠囊和耳蜗在光学透明胶原 i 凝胶保存固有的组织形态学, 允许机械刺激通过调整基质刚度, 并允许病毒介导的基因传递。

视频: Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

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 ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

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

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

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, ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

牙龈三维炎症人体组织当量

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...