Name: organotypic culture method to study the development of embryonic chicken tissues
Uploaded: 2018-08-25T12:30:00
Description: Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

作者要感谢格雷戈里 Haller (圣文森特大学) 在发展《议定书》方面的初步工作。作者还要感谢尼古拉斯. 琼斯 (圣文森特大学) 的技术专长和协助, 拍摄和制作手稿的视听部分。丹尼尔安德鲁斯得到了来自 MSVU 和加拿大自然科学和工程研究委员会 (NSERC) 的资助,通过了一项本科生研究奖。塔玛拉. Odendaal 是 NSERC 发现补助金的支持。

注: 对于胚胎阶段, 利用汉堡包和哈密尔顿12 (HH) 分期表。
1. 胚胎孵化
<ol><li>在37°c 1 °c 和40% 湿度的无菌、温度控制的孵化器中孵化受精的鸡卵。</li>
	<li>每天把鸡蛋 1x, 让它们孵化到 HH34 (受精后8天)。</li>
</ol>2. 材料的制备和灭菌
<ol><li>12胚, 高压釜2升蒸馏水, 1 升0.85% 鸡盐水, 12 玻璃吸管, 1 盒纸组织, 24 2.5 厘米 x 2.5 厘米的半多孔滤纸的?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+motor+proteins+in+echinoderm+embryos:+an+argument+in+support+of+antibody+inhibition+experiments.">Functions of motor proteins in echinoderm embryos: an argument in support of antibody inhibition experiments.</a> Cell Motility and the Cytoskeleton. 39 (4), 257-260 (1998).</li><li>Horakova, D., 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=Effect+of+FGFR+inhibitors+on+chicken+limb+development.">Effect of FGFR inhibitors on chicken limb development.</a> Development, Growth &amp; Differentiation. 56 (8), 555-572 (2014).</li><li>Gerlach, G. F., Morales, E. E., Wingert, R. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+organ+culture+of+embryonic+chick+femora:+a+system+for+investigating+bone+and+cartilage+formation+at+an+intermediate+level+of+organization.">Long-term organ culture of embryonic chick femora: a system for investigating bone and cartilage formation at an intermediate level of organization.</a> Journal of Bone and Mineral Research. 5 (1), 85-100 (1990).</li><li>Jung, H. 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=Local+inhibitory+action+of+BMPs+and+their+relationships+with+activators+in+feather+formation:+implications+for+periodic+patterning.">Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.</a> Developmental Biology. 196 (1), 11-23 (1998).</li><li>McKinnell, I. W., Turmaine, M., Patel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+Hedgehog+functions+by+localizing+the+region+of+proliferation+in+early+developing+feather+buds.">Sonic Hedgehog functions by localizing the region of proliferation in early developing feather buds.</a> Developmental Biology. 272 (1), 76-88 (2004).</li><li>Smith, E. L., Kanczler, J. M., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+take+on+an+old+story:+chick+limb+organ+culture+for+skeletal+niche+development+and+regenerative+medicine+evaluation.">A new take on an old story: chick limb organ culture for skeletal niche development and regenerative medicine evaluation.</a> European Cells &amp; Materials. 26, 91-106 (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+Spermatogenesis+in+Explanted+Adult+Mouse+Testis+Tissues.">In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.</a> PLoS One. 10 (6), e0130171 (2015).</li><li>Ochoa-Villarreal, 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=Plant+cell+culture+strategies+for+the+production+of+natural+products.">Plant cell culture strategies for the production of natural products.</a> BMB Reports. 49 (3), 149-158 (2016).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>Yu, 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=The+developmental+biology+of+feather+follicles.">The developmental biology of feather follicles.</a> The International Journal of Developmental Biology. 48, 181-191 (2004).</li><li>Jabalee, J., Hillier, S., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+cellular+dynamics+during+the+development+of+intramembranous+bones:+the+scleral+ossicles.">An investigation of cellular dynamics during the development of intramembranous bones: the scleral ossicles.</a> Journal of Anatomy. 223 (4), 311-320 (2013).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental Cell Research. 16 (1), 118-147 (1959).</li><li>Pinto, C. B., Hall, B. 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本协议利用已建立的组织培养技术, 从体外培养8 (HH34) 的胚胎4天, 实现鸡眼的生长。这种网格培养方法最初是由 Trowell15描述的。我们优化了一个协议, 从托比和霍尔的1991研究16利用半孔膜与网格, 研究诱导信号之间的分离组织层的胚胎鸡眼15。用这种方法, 蟑螂培养了14天的老鸡股骨6。这种方法也证明了有效的培养小鼠?...

胚胎鸡肉 (鸡鸡) 是生物学领域中常用的一种优良的模型生物。其潜伏期约为21天, 许多卵可以同时孵化, 使实验快速、高效。也许最重要的是 , 胚胎也很容易控 , 从而能够对关键的发育过程以及驱动这些过程的基因和蛋白质进行广泛的研究。
胚胎鸡眼是一个复杂的器官,通过与许多其他身体系统相似的不同组织的相互作用而发展。这种方法使研究这些组织的发展, 特别是在发展的先进阶段。例如, 多层视网膜可能对那些研究神经系统发育的人特别感兴趣。其他的方法, 使研究其他眼睛组织, 如角膜, vitreal 体, 晶状体, 巩膜, 和眼睑是有益的研究人员。鸡胚眼还含有一系列扁平骨, 即巩膜听骨, 可作为研究脊椎动物1膜内骨诱导和骨化的模型。
目前, 有许多方法用于研究胚胎发育。Microinjections 抑制性抗体或其他抑制分子2,3, 手术植入微球浸泡在抑制剂4, 和电穿孔5是所有的方法, 可用于 downregulate 基因或者是胚胎中感兴趣的蛋白质。类似的方法用于 upregulate 蛋白质。这些方法不是没有限制的。例如, 当使用化学物质来改变胚胎发育时, 必须对胚胎的致命影响进行评估, 这就限制了使用上述方法在足够低的剂量下对应用地点进行本地化, 以确保胚胎。
体外组织培养已被广泛应用于各种生物体中, 用于研究发展, 可用于绕过上述一些限制。例如, 股骨6、羽毛芽7、8和四肢9的鸡都用组织培养方法进行了研究, 如老鼠10的睾丸和植物的根和茎11。这些方法给予科学家高度的控制组织发展, 例如有能力波动的温度和改变养分的可用性。从整个胚胎中分离出的组织也使得它更不易受到化学物质的致命影响, 从而使得在全球范围内进行更高浓度的操纵研究。体外培养的另一个显著优点是保存组织的细胞环境;组织的排列保持相对不变, 从而可以研究不同组织类型9之间的相互作用。因此,在体外培养打开门的其他实验方法没有在体内或蛋模型。
目前, 使用化学物质研究胚胎鸡眼的发展尤其具有挑战性。许多胚外膜覆盖胚胎, 使其难以应用微球或化学物质;胚胎在卵子中也非常活跃, 因为它变老了, 使已经很难的方法复杂化了。该协议能够方便地接触眼睛及其周围组织, 消除这些障碍, 同时也为检查眼睛内的发育过程提供了新的机会。本协议的建立是为了研究在胚胎眼中的巩膜听骨的诱导。

<table border="0" cellpadding="0" cellspacing="0" style="width:1081px;" width="1082">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353001</td>
			<td style="width:676px;">easy grip tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td>tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper tissue</td>
			<td style="width:71px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Kimtech Science Brand Task Wipers, 280 per carton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">wire mesh</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>stainless steel wire mesh (grid size 0.7 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable glass pipettes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">14673-010</td>
			<td>Borosilicate glass disposable 5 3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nutrient medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">12591-038</td>
			<td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">penicillin-streptomycin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458</td>
			<td>10000 units/mL penicillin streptomycin solution stabilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1454 090</td>
			<td>semi-porous filter paper 90mm</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">fertilized chicken eggs</td>
			<td style="width:71px;">Dalhousie University Agricultural College</td>
			<td style="width:119px;">n/a</td>
			<td>can be obtained from local farms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride (NaCl)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0420-3</td>
			<td>sodium chloride crystals, reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 L glass bottle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">89000-240</td>
			<td>1 L pyrex autoclavable glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>BP82011</td>
			<td>70% molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tupperware containers</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable razor blades</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td>single edge industrial razor blades (surgical carbon steel)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic spoons</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dust mask</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">n/a</td>
			<td>3M 8500 Comfort Mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>paraformaldehyde, reagent grade, crystalline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neutral-buffered formalin</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">72210</td>
			<td>10% neutral buffered formalin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phosphate buffered saline (PBS)</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml falcon tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td>presterilized centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">n/a</td>
			<td>fine forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chick saline&#160;</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a&#160;</td>
			<td>0.85% NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tinfoil</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper towel</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
	</tbody>
</table>

organotypic culture method to study the development of embryonic chicken tissues

胚胎鸡通常被用作脊椎动物发育的可靠模型有机体。它的可达性和短潜伏期使其成为实验的理想选择。目前, 对鸡胚中这些发育途径的研究是通过在局部部位应用抑制剂和药物, 在低浓度下使用多种方法进行的。体外培养是一种技术, 它能够研究与宿主生物体分离的组织, 同时绕过与整个胚胎工作时存在的许多物理限制, 如胚胎的易感性高剂量的潜在致命化学物质。在这里, 我们提出了一个脊髓培养的方法, 以培养胚胎鸡半头的体外, 这为检查发展过程的新的机会, 超越目前建立的办法。

采用这种方法, 可以在体外培养8天的胚胎鸡眼 (HH34), 4 天。四天的蛋发展对应于 HH38。
这种培养方法支持发展的羽毛芽周围的眼睛和眼睑 (图 1B)。这些羽毛芽不存在于蛋在 HH34 之前的培养 (图 1A), 表明羽毛芽发育是在培养期间开始, 因为它是在蛋<sup class="xre...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

脊髓培养法研究胚胎鸡组织的发展

在这里, 我们提出了一个脊髓培养协议, 培育胚胎鸡器官体外.利用这种方法, 可以对胚胎鸡组织的发育进行研究, 同时保持对培养环境的高度控制。

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Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

一本小说<em&gt;防爆大毛</em&gt;带技术来改变心内血流动力学在鸡胚胎系统

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

小鼠胚胎跖骨文化：软骨内骨化的生理模型

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

胸腺标本分离和鸡刺激器官的形成

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...