这项工作得到了美国国立卫生研究院（NIH）资助：RO1 EY012085（给JXJ）和F32DK134051（给FMA）以及韦尔奇基金会资助：AQ-1507（给JXJ）。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。这些数字部分是用 Biorender.com 创建的。

本研究是按照《动物福利法》和《动物福利实施条例》的原则，根据《实验动物护理和使用指南》的原则进行的。所有动物程序均由德克萨斯大学圣安东尼奥健康科学中心机构动物护理和使用委员会批准。有关协议的概述，请参阅 图 1;有关本协议中使用的所有材料、试剂和仪器的详细信息，请参阅 材料表 。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65727/65727fig01.jpg"> 图1：实验大纲。1. 方案的第一步是确定特定的靶蛋白，鉴定相关基因序列，并生成 DNA 片段。 2 . 通过初始克隆到接头载体中，将基因序列克隆到逆转录病毒载体中， 3. 然后是病毒载体。 4. 使用包装细胞制备高滴度病毒颗粒，以收获和浓缩。 5. 最后一步，也是该协议的重点，是将RCAS（A）病毒颗粒的鸡晶状体显微注射到晶状体腔中。 <a href="https://www.jove.com/files/ftp_upload/65727/65727fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
1. 高滴度重组逆转录病毒的制备
<ol><li>聚合酶链反应 （PCR） 制备靶蛋白 DNA 片段 注：本节旨在扩增与靶晶状体蛋白相对应的DNA序列。有关详细信息，请参见 7,8。<ol><li>设计与目标蛋白相对应的PCR引物，以制备具有羧基末端的DNA片段，具有或不具有FLAG表位序列（5'-GACTACAAGGACGACGATGACAAG-3'），终止密码子和限制性内切酶位点（即EcoRI）存在于CLa12NCO的多接头区域内）根据先前发表的方案中注明的规格，具有足够的限制性内切酶的序列1，7、8、10、11。</li>
		<li>进行PCR反应10。将设计引物的 100 pmol 正义和反义与 0.5 μg 连接蛋白 cDNA（例如 Cx50、Cx43 或嵌合 Cx50*43L 组合）混合到所选的 PCR 反应缓冲液中。执行30个循环，每个循环包括94°C1分钟，58°C1分钟和72°C1分钟，然后在72°C下最终延伸10分钟。</li>
		<li>使用凝胶纯化试剂盒分离和纯化PCR产物。</li>
		<li>根据制造商的说明，使用选择的限制性内切酶消化纯化的PCR产物。</li>
	</ol></li>
	<li>将DNA插入片段克隆到接头质粒中 注：将 DNA 片段插入接头载体中无需辅助细胞/病毒即可提高 RCAS（A） 载体稳定性14.有关详细信息，请参见 7,8。<ol><li>使用粘性末端连接反应将 DNA 片段亚克隆到衔接质粒 Cla12NCO 中。将PCR片段与限制性内切酶和Cla12NCO以2-5：1的比例混合。使用感受态细胞进行连接和DNA转化。</li>
		<li>使用 DNA 分离试剂盒分离 DNA。</li>
		<li>对 DNA 进行测序以确保准确性。</li>
	</ol></li>
	<li>克隆到RCAS（A）病毒载体中 注：将DNA片段插入载体（RCAS（A）逆转录病毒）中，以便将基因稳定转导到发育中的鸡胚14,15的细胞中。有关详细信息，请参见7、8、15。<ol><li>用 ClaI（每 1 μg DNA 1 个单位）消化目的载体的 Cla12NCO-DNA 片段并凝胶分离片段。</li>
		<li>将 DNA 片段亚克隆到 ClaI 线性化的 RCAS（A） 质粒中。使用限制性内切酶SalI区分RCAS（A）质粒上的ClaI位点。</li>
		<li>通过用 ClaI 和 SalI 限制性内切酶消化来确认插入片段在构建体上的正确方向。</li>
		<li>使用 DNA 分离试剂盒分离 DNA。</li>
		<li>测定DNA浓度。</li>
	</ol></li>
	<li>雏鸡胚胎成纤维细胞 （CEF） 细胞的转染和 RCAS（A） 收获 注：病毒包装细胞 CEF 用于获取/收获含有病毒颗粒的细胞培养基15,16。有关详细信息，请参见7、8、15。<ol><li>根据制造商的说明，使用转染试剂用重组逆转录病毒 DNA 构建体转染 CEF 细胞。</li>
		<li>对转染细胞进行蛋白质印迹，以检查其目标蛋白的表达。</li>
		<li>当转染的细胞达到汇合时，开始收集上清液培养基，对其进行适当的处理/过滤（使用0.22μm过滤器去除任何细胞碎片），并储存在-80°C直至准备浓缩。</li>
	</ol></li>
	<li>病毒储备液的浓度和滴度 注意：必须过滤来自含有病毒颗粒的细胞的沉淀和大量培养基。此外，必须进行病毒滴度测定以确定病毒对宿主细胞的强度。有关详细信息，请参见6、7、8、15。<ol><li>解冻含有病毒粒子的培养基。</li>
		<li>将培养基在4°C下以72,000× g 旋转2小时。</li>
		<li>倒出上清液，用残留的（~50μL）培养基重悬病毒沉淀，并在-80°C下储存在~10μL病毒储备液中直至使用。</li>
		<li>对于滴度测定，使用 QT-6 或 CEF 细胞，用病毒储备液的连续稀释液转染细胞。</li>
		<li>汇合后，使用4%甲醛固定细胞。</li>
		<li>病毒 gag 蛋白的免疫染色或碱性磷酸酶的组织化学过程以获得滴度，定义为（集落形成单位）CFU/毫升 （CFU/mL）。</li>
	</ol></li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65727/65727fig02.jpg"> 图 2：雏鸡晶状体显微注射的仪器和设置 。 （A） P-30手动立式微电极微量移液器拉拔器。（1） 插图显示正在拉动的玻璃微量移液器。（B） （2） 种蛋孵化器。将鸡蛋在37°C培养箱中孵育~65-68小时，以达到注射逆转录病毒的第18阶段（具有密封的空中央腔）。（C） 显微注射设置。（3）照明设备，（4）微型注射器，（5）解剖显微镜，（6）德拉蒙德显微操纵器，（7）用于可视化的计算机/相机。 <a href="https://www.jove.com/files/ftp_upload/65727/65727fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
2. 小鸡晶状体显微注射
<ol><li>准备用品<ol><li>显微注射用玻璃微量移液器的制备17 注：玻璃毛细管用于制造尖端点和外径 （OD） 为 ~11 μm 的玻璃微量移液器，用于显微注射。设置如图 2A所示。<ol><li>将硼硅酸盐玻璃毛细管连接到手动立式微量移液器拉拔器中的橡胶缓冲夹上。</li>
			<li>将加热温度 （HEAT 1） 设置为 950 °C 并进行预拉以获得更薄、更柔软的玻璃毛细管。</li>
			<li>将加热温度（HEAT 2）设置为790°C，并进行二次拉动以产生最终的微量移液器。</li>
			<li>使用微量移液器研磨机，将吸头开口锐化至外径~11μm。</li>
			<li>对于未来的实验，将玻璃微量移液器放在玻璃罐内的海绵夹紧垫中。</li>
		</ol></li>
		<li>受精卵孵化至发育阶段 18 注：在晶状体发育过程中，在胚胎发育的~65-68小时，晶状体与外胚层分离并形成具有中央腔的密封囊泡;注射到这个空晶状体腔中，限制表达到增殖的晶状体细胞7,8,18。<ol><li>将受精鸡蛋在37°C加湿摇摆培养箱中孵育~65-68小时（图2B）。</li>
		</ol></li>
	</ol></li>
	<li>鸡蛋的打开 注意：此步骤描述了用于显微注射的胚胎的识别和暴露。有关详细信息，请参见 7,8。<ol><li>用 70% 乙醇彻底擦拭工作区域。</li>
		<li>从孵化器中取出鸡蛋，用 70% 乙醇大量喷洒，然后让它们风干。</li>
		<li>将鸡蛋的较大一端朝上放在蛋架上。</li>
		<li>使用一对锋利的齿镊子，小心地敲击蛋壳并去除蛋壳的碎片，在鸡蛋的较大端产生一个直径为~2厘米的孔（图3A）。</li>
		<li>使用解剖显微镜，定位胚胎。</li>
		<li>一旦找到胚胎和雏鸡晶状体，使用解剖显微镜和精细解剖镊子和剪刀切断立即覆盖胚胎顶部的羊膜（图3B）。 注意：不要切得太宽（仅足以暴露胚胎~0.5 cm x 0.5 cm），否则胚胎可能会沉入卵黄块中;如果可能的话，也要避免接触血管。</li>
		<li>用60毫米的培养皿盖住鸡蛋的开口，为下一步做准备。</li>
	</ol></li>
	<li>显微注射浓缩病毒储备液 注：将含有用于转导的靶蛋白DNA片段的病毒颗粒插入晶状体腔内的细胞中。设置如图 2C 所示。有关详细信息，请参见6、7、8。<ol><li>在冰上解冻病毒库存。</li>
		<li>为了在注射过程中可视化病毒原液，将 1 μL 的 10x Fast Green 稀释到一小瓶含有 10 μL 的病毒原液中。</li>
		<li>为确保没有大块未溶解的物质可能堵塞玻璃微量移液器，在4°C下以10,000× g 离心溶液10秒，以沉淀“大块”并将上清液转移到新管中，同时将溶液保持在冰上。</li>
		<li>在 35 mm x 10 mm 培养皿上，放置拉伸的实验室封口膜，并在薄膜顶部加入 1 μL 无菌盐水 （PBS）（未灭菌，覆盖面被认为是“干净的”）。</li>
		<li>将准备好的玻璃微量移液器连接到自动微微注射器。</li>
		<li>按下 填充模式 ，将无菌盐水 （PBS） 填充到玻璃微量移液器中，然后使用 注射模式 测试分配的 1 μL 液体。</li>
		<li>将第二个拉伸的实验室封口膜放在新的 35 mm x 10 mm 细胞培养皿的表面上，并在薄膜顶部加入 1 μL 快速绿色染色的病毒储备液。</li>
		<li>将微量移液器的尖端降低到病毒储液中，然后按 压填充模型 ，用~1μL病毒储液器填充玻璃微量移液器。 注意：为避免变干，每当程序暂停时，将装满的微量移液器的尖端放入无菌盐水 （PBS） 中。</li>
		<li>在解剖显微镜的帮助下，将连接到自动注射器的填充玻璃微量移液器降低到胚胎晶状体腔的目标区域。</li>
		<li>调整光源以确保晶状体囊泡轮廓清晰可见。 注意：右晶状体的内腔（朝上）通常用于注射，左晶状体（朝下）保持完整作为对侧对照。</li>
		<li>一旦确定微量移液器的位置在晶状体腔内的正确位置，注射5-40nL的病毒储备液（图3C）。 注意：练习对于最佳注射位置非常必要。</li>
		<li>等待~45秒，然后轻轻取出玻璃微量移液器。</li>
		<li>使用解剖显微镜检查是否成功显微注射到晶状体腔中，方法是检查应保持在晶状体空腔中且没有任何泄漏的固绿染料。</li>
		<li>注射程序后，用透明胶带密封蛋壳开口。</li>
		<li>将胚胎放回37°C加湿的培养箱中，没有任何旋转运动，直到达到所需的胚胎年龄进行晶状体解剖。</li>
	</ol></li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65727/65727fig03.jpg"> 图 3：显微注射雏鸡制备和示意图 。 （A） 打开鸡蛋。（B）切割羊膜。（C）鸡晶状体腔显微注射示意图。 <a href="https://www.jove.com/files/ftp_upload/65727/65727fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

研究鸡晶状体发育过程中的蛋白质表达

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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> Developmental Dynamics. 195 (4), 231-272 (1992).</li><li>Hallagan, J. B., Allen, D. C., Borzelleca, J. 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+safety+and+regulatory+status+of+food,+drug+and+cosmetics+colour+additives+exempt+from+certification.">The safety and regulatory status of food, drug and cosmetics colour additives exempt from certification.</a> Food and Chemical Toxicology. 33 (6), 515-528 (1995).</li><li>Okada, T. S., Eguchi, G., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+differentiation+by+chicken+lens+epithelium+in+in+vitro+cell+culture.">The expression of differentiation by chicken lens epithelium in in vitro cell culture.</a> Development, Growth &amp; Differentiation. 13 (4), 323-336 (1971).</li><li>Menko, A. S., Klukas, K. A., Johnson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+embryo+lens+cultures+mimic+differentiation+in+the+lens.">Chicken embryo lens cultures mimic differentiation in the lens.</a> Developmental Biology. 103 (1), 129-141 (1984).</li><li>Parreno, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+to+unlock+the+molecular+expression+and+cellular+structure+of+ocular+lens+epithelial+cells.">Methodologies to unlock the molecular expression and cellular structure of ocular lens epithelial cells.</a> Frontiers in Cell and Developmental Biology. 10, 983178 (2022).</li><li>Edwards, A., Gupta, J. D., Harley, 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=Photomicrographic+evaluation+of+drug-induced+cataracts+in+cultured+embryonic+chick+lens.">Photomicrographic evaluation of drug-induced cataracts in cultured embryonic chick lens.</a> Experimental Eye Research. 15 (4), 495-498 (1973).</li><li>Musil, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+embryonic+chick+lens+cells+as+a+model+system+to+study+lens+gap+junctions+and+fiber+cell+differentiation.">Primary cultures of embryonic chick lens cells as a model system to study lens gap junctions and fiber cell differentiation.</a> Journal of Membrane Biology. 245 (7), 357-368 (2012).</li><li>West-Mays, J. A., Pino, G., Lovicu, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+the+lens+epithelial+explant+system+to+study+lens+differentiation+and+cataractogenesis.">Development and use of the lens epithelial explant system to study lens differentiation and cataractogenesis.</a> Progress in Retinal and Eye Research. 29 (2), 135-143 (2010).</li><li>Upreti, 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=Lens+epithelial+explants+treated+with+vitreous+humor+undergo+alterations+in+chromatin+landscape+with+concurrent+activation+of+genes+associated+with+fiber+cell+differentiation+and+innate+immune+response.">Lens epithelial explants treated with vitreous humor undergo alterations in chromatin landscape with concurrent activation of genes associated with fiber cell differentiation and innate immune response.</a> Cells. 12 (3), 501 (2023).</li><li>Walker, J. L., Wolff, I. M., Zhang, L., Menko, 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=Activation+of+SRC+kinases+signals+induction+of+posterior+capsule+opacification.">Activation of SRC kinases signals induction of posterior capsule opacification.</a> Investigative Ophthalmology &amp; Visual Science. 48 (5), 2214-2223 (2007).</li><li>Briskin, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+retroviral+transgenes+are+highly+expressed+in+chickens.">Heritable retroviral transgenes are highly expressed in chickens.</a> Proceedings of the National Academy of Sciences of the United States of America. 88 (5), 1736-1740 (1991).</li><li>Hughes, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCAS+vector+system.">The RCAS vector system.</a> Folia Biologica. 50 (3-4), 107-119 (2004).</li></ol>

作者声明，该研究是在没有任何可被解释为潜在利益冲突的商业或财务关系的情况下进行的。

该实验模型提供了在完整晶状体中表达目标蛋白质的机会，从而研究了这些蛋白质在晶状体结构和功能中的功能相关性。胚胎雏鸡显微注射模型部分基于Fekete等人的工作。al.6，并由Jiang等人进一步发展。al.8 并已被用作将病毒质粒和试剂（如激动剂、小干扰 RNA （siRNA） 和肽）插入雏鸡晶状体的手段 1,9,10,11,12,13。<...

该协议的目的是描述RCAS（A）（具有复制能力的禽肉瘤/白血病逆转录病毒A）的胚胎鸡晶状体显微注射的方法。在胚胎鸡晶状体中有效的逆转录病毒递送已被证明是 体内研究晶 状体蛋白在正常晶状体生理学、病理状况和发育中的分子机制和结构功能的有前途的工具。此外，该实验模型还可用于人类先天性白内障等疾病的治疗靶点识别和药物筛选。总而言之，该协议旨在为开发用于研究晶状体蛋白的可定制平台制定必要的步骤。
胚胎雏鸡（Gallus domesticus）由于其晶状体结构和功能与人类晶状体相似，是研究晶状体发育和生理学的成熟动物模型1,2,3,4。使用具有复制能力的 RCAS（A） 禽逆转录病毒被认为是在雏鸡胚胎中表达外源蛋白的高效、快速和可定制的系统。值得注意的是，它具有独特的能力，可以将靶基因转移限制在增殖的晶状体纤维细胞中，而不需要组织特异性启动子，使用独特的胚胎发育时间框架，其中空晶状体腔的存在允许原位 RCAS（A） 显微注射到受限位点，以便在增殖晶状体纤维细胞内表达外源性蛋白质5，6,7,8.
这里深入描述的鸡胚显微注射程序最初部分基于 Fekete 等人的工作。al.6 并由 Jiang 等人进一步发展。Al.8，并已被用作将病毒和非病毒质粒引入胚胎雏鸡晶状体的手段1,9,10,11,12,13。总体而言，先前的工作证明了利用这种方法研究晶状体发育、分化、细胞通讯和疾病进展的潜力，以及发现和测试晶状体病理状况（如白内障）的治疗靶点。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Filter</td>
			<td>Corning</td>
			<td>431118</td>
			<td>For removing cellular debris from media</td>
		</tr>
		<tr>
			<td>35 mm x 10 mm Culture Dish</td>
			<td>FisherScientific</td>
			<td>50-202-030</td>
			<td>For using during microinjection</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisherbrand</td>
			<td>13-100-676</td>
			<td>Spinning down solution</td>
		</tr>
		<tr>
			<td>Constructs</td>
			<td>GENEWIZ</td>
			<td>-</td>
			<td>For generation of constructs</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>AmScope</td>
			<td>SM-4TZ-144A</td>
			<td>Visualization of lens for microinjection</td>
		</tr>
		<tr>
			<td>DNA PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td>-</td>
			<td>Generation of primers: 
			 
			Intracellular loop (IL)-deleted Cx50 (residues 1&#8211;97 and 149&#8211;400) as well as the Cla12NCO vector were obtained with the following pair of primers: sense, CTCCTGAGAACCTACATCCT; antisense, CACCGCATGCCCAAAGTACAC 
			 
			ILs of Cx43 (residues 98&#8211;150) and Cx46 (residues 98&#8211;166) were&#160; obtained with the following pairs of primers: sense, TACGTGATGAGGAAAGAAGAG; antisense, TCCTCCACGCATCTTTACCTTG; sense, CACATTGTACGCATGGAAGAG; antisense, AGCACCTCCC AT ACGGATTC, respectively 
			 
			Cla12NCO-Cx43 construct template was obtained with the following pair of primers: sense, CTGCTTCGTACTTACATCATC; antisense, GAACAC GTGCGCCAGGTAC 
			 
			ILs of Cx50 (residues 98&#8211;148) or Cx46 (residues 98&#8211;166) were cloned by using Cla12NCO-Cx50 and Cla12NCO-Cx46 constructs as the templates with the following pair of primers: sense, CACCATGTCCGCATGGAGGAGA; antisense, GGTCCCC TC CAGGCGAAAC; sense, CACATTGTACGCATGGAAGAG; antisense, AGCACCTCCCATACGGATTC, respectively</td>
		</tr>
		<tr>
			<td>Drummond Nanoject II Automatic Nanoliter Injector</td>
			<td>Drummond Scientific</td>
			<td>3-000-204</td>
			<td>Microinjection Pipet</td>
		</tr>
		<tr>
			<td>Dual Gooseneck Lights Microscope Illuminator</td>
			<td>AmScope</td>
			<td>LED-50WY</td>
			<td>Lighting for visualization</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Egg Holder</td>
			<td>-</td>
			<td>-</td>
			<td>Homemade styrofoam rings with 2-inch diameter and one-half inch height</td>
		</tr>
		<tr>
			<td>Egg Incubator</td>
			<td>GQF Manufacturing Company Inc.</td>
			<td>1502</td>
			<td>For incubation of fertilized eggs</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fisher scientific</td>
			<td>F99-10</td>
			<td>For visualization of viral stock injection</td>
		</tr>
		<tr>
			<td>Fertilized white leghorn chicken eggs</td>
			<td>Texas A&#38;M University</td>
			<td>N/A</td>
			<td>Animal model of choice for microinjection (https://posc.tamu.edu/fertile-egg-orders/)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone Laboratories</td>
			<td></td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Fluorescein-conjugated anti-mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-095-003</td>
			<td>For anti-FLAG&#160; 1:500</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FisherScientific</td>
			<td>22-327379</td>
			<td>For moving things around and isolation</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Sutter Instruments</td>
			<td>B100-75-10</td>
			<td>Glass micropipette for microinjection (O.D. 1.0 mm, I.D. 0.75 mm, 10 cm length)</td>
		</tr>
		<tr>
			<td>Lipofectamine</td>
			<td>Invitrogen</td>
			<td>L3000001</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>Manual vertical micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-30</td>
			<td>To obtain glass micropipette of the correct size</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>FisherScientific</td>
			<td>02-682-004</td>
			<td>Dissolving solution</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td>For imaging staining</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>FisherScientific</td>
			<td>03-448-254</td>
			<td>Placing solution</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Pico-Injector</td>
			<td>Harvard Apparatus</td>
			<td>PLI-100</td>
			<td>For delivering small liquid volumes precisely through micropipettes by applying a regulated pressure for a digitally set period of time</td>
		</tr>
		<tr>
			<td>rabbit anti-chick AQP0</td>
			<td>Self generated</td>
			<td>-</td>
			<td>Jiang JX, White TW, Goodenough DA, Paul DL. Molecular cloning and functional characterization of chick lens fiber connexin 45.6. Mol Biol Cell. 1994 Mar;5(3):363-73. doi: 10.1091/mbc.5.3.363.</td>
		</tr>
		<tr>
			<td>rabbit anti-FLAG antibody</td>
			<td>Rockland Immunichemicals</td>
			<td>600-401-383</td>
			<td>For staining FLAG</td>
		</tr>
		<tr>
			<td>Rhodamine-conjugated anti-rabbit IgG&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-295-003</td>
			<td>For anti-AQP0&#160; 1:500</td>
		</tr>
		<tr>
			<td>Sponge clamping pad</td>
			<td>Sutter Instruments</td>
			<td>BX10</td>
			<td>For storage of glass micropipette</td>
		</tr>
	</tbody>
</table>

microinjection of recombinant rcas(a) retrovirus into embryonic chicken lens

胚胎鸡（Gallus domesticus）是一种成熟的动物模型，用于研究晶状体发育和生理学，因为它与人类晶状体高度相似。RCAS（A）是一种具有复制能力的鸡逆转录病毒，可感染分裂细胞，是研究晶状体发育过程中野生型和突变蛋白原 位 表达和功能的有力工具，在早期发育阶段通过显微注射到晶状体囊泡的空腔中，将其作用限制在周围增殖的晶状体细胞中。与转基因模型和 离体 培养等其他方法相比，使用具有复制能力的禽逆转录病毒提供了一种高效、快速和可定制的系统来表达雏鸡胚胎中的外源蛋白。具体来说，靶向基因转移可以局限于增殖的晶状体纤维细胞，而不需要组织特异性启动子。在本文中，我们将简要概述重组逆转录病毒RCAS（A）制备所需的步骤，提供显微注射程序的详细、全面的概述，并提供该技术的样本结果。

The goal of this protocol is to describe the methodology of embryonic chicken lens microinjection of an RCAS(A) (replication-competent avian sarcoma/leukosis retrovirus A). Effective retroviral delivery in an embryonic chicken lens has been demonstrated to be a promising tool for the in vivo study of the molecular mechanism and structure-function of lens proteins in normal lens physiology, pathological conditions, and development. Moreover, this experimental model could be used for the identification of therapeutic targets and drug screening for conditions such as human congenital cataracts. In all, this protocol aims to lay out the necessary steps for the development of a customizable platform for the study of lens proteins.
Embryonic chicks (Gallus domesticus), owing to their similarity in lens structure and function with the human lens, are a well-established animal model for the study of lens development and physiology1,2,3,4. The use of an RCAS(A) replication-competent avian retrovirus has been regarded as a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Notably, it has a unique ability to confine the target gene transfer to proliferative lens fiber cells without the need for tissue-specific promoters, using the unique embryonic development time frame in which the presence of empty lens lumen permits in situ RCAS(A) microinjection into the restricted site for the expression of exogenous proteins within proliferative lens fiber cells5,6,7,8.
The chick embryo microinjection procedure, described in-depth here, is based originally partially on the work of Fekete et. al.6 and further developed by Jiang et. al.8 and has been utilized as a means of introducing both viral and nonviral plasmids into the lens of embryonic chicks1,9,10,11,12,13. Overall, the previous work demonstrates the potential of utilizing this methodology to study lens development, differentiation, cellular communication, and disease progression, and for the discovery and testing of therapeutic targets for lens pathological conditions such as cataracts.

作者聚焦：通过在胚胎鸡晶状体中显微注射 RCAS（A） 逆转录病毒来研究晶状体的发育和功能 

重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中

The presented methodology of embryonic chicken lens microinjection of a RCAS retrovirus is meant as a tool for studying in situ function and expression of proteins during lens development. Compared to other approaches, such as transgenic models and ex vivo cultures, the RCAS replication-competent avian retrovirus provides a highly-effective, rapid, and customizable system for expressing exogenous proteins in chick embryos. Targeted gene transfer can be confined to proliferative lens fiber cells without the need for promoters.There is prominent potential for utilizing this methodology to study lens development, differentiation, cellular communication, and disease progression, and for the discovery and testing of therapeutic targets for lens pathological conditions such as cataracts.

在确定特定靶蛋白和鉴定相关基因序列后，总体实验方法包括将基因序列克隆到逆转录病毒 RCAS（A） 载体中，方法是初始克隆到接头载体中，然后是病毒载体。其次，使用包装细胞制备高滴度病毒颗粒，以收获和浓缩病毒粒子。前两个主要步骤已基本描述，代表性结果在其他地方呈现6、7、8、14、<sup...

Watch this Scientific Journal Video about Investigating Lens Development and Function Through Microinjection of RCASA Retrovirus in Embryonic Chicken Lens at JoVE.com

该协议论文描述了RCAS（A）逆转录病毒的胚胎鸡晶状体显微注射的方法，作为研究晶状体发育过程中蛋白质原 位 功能和表达的工具。

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...

所提出的RCAS逆转录病毒胚胎鸡晶状体显微注射方法旨在作为研究晶状体发育过程中蛋白质原位功能和表达的工具。与转基因模型和离体培养等其他方法相比，具有复制能力的禽逆转录病毒为在雏鸡胚胎中表达外源蛋白提供了一种高效、快速和可定制的系统。靶向基因转移可以局限于增殖的晶状体纤维细胞，而不需要启动子。利用这种方法研究晶状体发育、分化、细胞通讯和疾病进展，以及发现和测试晶状体病理状况（如白内障）的治疗靶点具有突出的潜力。

microinjection-recombinant-rcasa-retrovirus-into-embryonic-chicken

Research

JoVE Journal

Biology

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

699 次观看.  University of Texas Health Science Center, San Antonio. 所提出的RCAS逆转录病毒胚胎鸡晶状体显微注射方法旨在作为研究晶状体发育过程中蛋白质原位功能和表达的工具。与转基因模型和离体培养等其他方法相比，具有复制能力的禽逆转录病毒为在雏鸡胚胎中表达外源蛋白提供了一种高效、快速和可定制的系统。靶向基因转移可以局限于增殖的晶状体纤维细胞，而不需要启动子。利用这种方法研究晶状体发育、分化、细胞通...

视频: 作者聚焦：通过在胚胎鸡晶状体中显微注射 RCAS（A） 逆转录病毒来研究晶状体的发育和功能 

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

科研

生物学

Preparation of Chicken Lens for Microinjection of an RCAS(A) Retrovirus

To begin, attach the borosilicate glass capillary to the rubber cushioned clips in the manual vertical micropipette puller. Set the heat temperature of the micropipette puller to 950 degrees Celsius and perform pre-pulling to obtain a thinner and softer glass capillary. Then set the heat temperature to 790 degrees Celsius and perform a secondary pulling to produce final micropipettes.Using a micropipette grinder, carefully sharpen the tip opening to an outer diameter of 11 micrometers. Then place the micropipettes in a sponge clamping pad inside of a glass jar. Incubate the fertilized chicken eggs until development stage 18 in a 37 degree Celsius humidified rocking incubator.Wipe down the working area thoroughly with 70%ethanol. After development, remove the eggs from the incubator. Spray the eggs with 70%ethanol and allow them to air dry.Then place the egg on an egg holder with the larger end facing upwards. Using a pair of sharp toothed forceps, carefully tap on the larger end of the eggshell to create a two centimeter diameter hole. Then remove the fragments of the egg shell.Place the egg on a dissecting microscope stage and locate the embryo. Next, use the fine dissecting forceps and scissors to cut off the amniotic membrane covering the top of the embryo. Then place a 60 millimeter Petri dish over the opening of the egg.

用于显微注射RCAS（A）逆转录病毒的鸡晶状体的制备

Microinjection of an RCAS(A) Retrovirus into Embryonic Chicken Lens for Expressing Exogenous Proteins 

To begin, thaw the RCAS(A)retrovirus stock on ice. To avoid clogging the micropipette, centrifuge the solution at 10, 000 G for 10 seconds at four degrees Celsius and transfer the supernatant into a new tube while keeping the solutions on ice. Place a stretched laboratory parafilm on a 35 by 10 millimeter cell culture dish and add one microliter of sterile PBS to the parafilm.Connect a prepared glass micropipette to an automatic Pico injector. Press the fill mode to fill the PBS into a micropipette and then press the inject mode to test the allocated one microliter of liquid. Place a second piece of stretched laboratory parafilm on a new cell culture dish and add one microliter of fast green stained viral stock to the film.Lower the tip of the micropipette into the viral stock, and press the fill mode to fill the micropipette with one microliter of viral stock. Under a dissecting microscope, lower the filled micropipette connected to an automatic injector into the target region of the chicken embryo lens. Then adjust the light source to provide a clear view of the lens vesicle.After ensuring the micropipette is within the correct location inside the lens lumen, inject five to 40 nanoliters of the viral stock. After injection, wait 45 seconds before gently removing the micropipette. Next, check the successful microinjection by examining the fast green dye in the empty lumen of the lens without an leakage.Following examination, seal the eggshell opening with scotch tape and place the eggs in a 37 degree Celsius humidified static incubator. This study demonstrates the histological evaluation of the chimeric connexon 50 and 43 loops through immunofluorescence. Using Anti-Flag labeling, exogenous connexons were distinguished from endogenous ones.The study also evaluates the interaction between the intracellular loop domain of connexon 50 and Aquaporin zero.

作者聚焦：通过在胚胎鸡晶状体中显微注射 RCAS（A） 逆转录病毒来研究晶状体的发育和功能

作者聚焦：通过在胚胎鸡晶状体中显微注射 RCAS（A）逆转录病毒来研究晶状体的发育和功能