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

<ol>
	<li>Li, Z., Gu, S., Quan, Y., Varadaraj, K., Jiang, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+potent+embryonic+chick+lens+model+for+studying+congenital+cataracts+in+vivo.">Development of a potent embryonic chick lens model for studying congenital cataracts in vivo.</a> Communications Biology. 4 (1), 325 (2021).</li><li>Chen, Y., 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=&#947;-Crystallins+of+the+chicken+lens:+remnants+of+an+ancient+vertebrate+gene+family+in+birds.">&#947;-Crystallins of the chicken lens: remnants of an ancient vertebrate gene family in birds.</a> The FEBS Journal. 283 (8), 1516-1530 (2016).</li><li>Coulombre, A. J., Coulombre, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+development.+I.+Role+of+the+lens+in+eye+growth.">Lens development. I. Role of the lens in eye growth.</a> Journal of Experimental Zoology. 156 (1), 39-47 (1964).</li><li>McKeehan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+portions+of+the+chick+lens+without+contact+with+the+optic+cup.">Induction of portions of the chick lens without contact with the optic cup.</a> The Anatomical Record. 132 (3), 297-305 (1958).</li><li>Kothlow, S., Schenk-Weibhauser, K., Ratcliffe, M. J., Kaspers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+effect+of+BAFF+on+chicken+B+cell+development+revealed+by+RCAS+retroviral+gene+transfer+in+vivo.">Prolonged effect of BAFF on chicken B cell development revealed by RCAS retroviral gene transfer in vivo.</a> Molecular immunology. 47 (7-8), 1619-1628 (2010).</li><li>Fekete, D. M., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-competent+retroviral+vectors+encoding+alkaline+phosphatase+reveal+spatial+restriction+of+viral+gene+expression/transduction+in+the+chick+embryo.">Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo.</a> Molecular and Cellular Biology. 13 (4), 2604-2613 (1993).</li><li>Jiang, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+retroviruses+to+express+connexins.">Use of retroviruses to express connexins.</a> Methods in Molecular Biology. , 159-174 (2001).</li><li>Jiang, J. X., Goodenough, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+expression+of+connexins+in+embryonic+chick+lens.">Retroviral expression of connexins in embryonic chick lens.</a> Investigative Ophthalmology &amp; Visual Science. 39 (3), 537-543 (1998).</li><li>Shestopalov, V. I., Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+autofluorescent+proteins+reveals+a+novel+protein+permeable+pathway+between+cells+in+the+lens+core.">Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core.</a> Journal of Cell Science. 113 (11), 1913-1921 (2000).</li><li>Liu, J., Xu, J., Gu, S., Nicholson, B. J., Jiang, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquaporin+0+enhances+gap+junction+coupling+via+its+cell+adhesion+function+and+interaction+with+connexin+50.">Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50.</a> Journal of Cell Science. 124 (2), 198-206 (2011).</li><li>Li, Z., 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+second+extracellular+domain+of+connexin+50+is+important+for+in+cell+adhesion,+lens+differentiation,+and+adhesion+molecule+expression.">The second extracellular domain of connexin 50 is important for in cell adhesion, lens differentiation, and adhesion molecule expression.</a> Journal of Biological Chemistry. 299 (3), 102965 (2023).</li><li>Shestopalov, V. I., Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+gene+expression+and+protein+targeting+in+lens+fiber+cells.">Exogenous gene expression and protein targeting in lens fiber cells.</a> Investigative Ophthalmology &amp; Visual Science. 40 (7), 1435-1443 (1999).</li><li>Shestopalov, V. I., Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organization+of+primary+lens+fiber+cells.">Three-dimensional organization of primary lens fiber cells.</a> Investigative Ophthalmology &amp; Visual Science. 41 (3), 859-863 (2000).</li><li>Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J., Sutrave, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptor+plasmids+simplify+the+insertion+of+foreign+DNA+into+helper-independent+retroviral+vectors.">Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors.</a> Journal of Virology. 61 (10), 3004-3012 (1987).</li><li>Yan, R. T., Wang, S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+RCAS+retrovirus.">Production of high-titer RCAS retrovirus.</a> Methods in Molecular Biology. 884, 193-199 (2012).</li><li>Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+mammalian+cells.">Introduction of DNA into mammalian cells.</a> Current Protocols in Molecular Biology. 64 (1), 1-95 (2003).</li><li>Li, Y., 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=Studying+macrophage+activation+in+immune-privileged+lens+through+CSF-1+protein+intravitreal+injection+in+mouse+model.">Studying macrophage activation in immune-privileged lens through CSF-1 protein intravitreal injection in mouse model.</a> STAR Protocols. 3 (1), 101060 (2022).</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> 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><tbody><tr><td>0.22 &amp;micro;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:&lt;br/&gt; &lt;br/&gt; Intracellular loop (IL)-deleted Cx50 (residues 1&amp;ndash;97 and 149&amp;ndash;400) as well as the Cla12NCO vector were obtained with the following pair of primers: sense, CTCCTGAGAACCTACATCCT; antisense, CACCGCATGCCCAAAGTACAC&lt;br/&gt; &lt;br/&gt; ILs of Cx43 (residues 98&amp;ndash;150) and Cx46 (residues 98&amp;ndash;166) were&amp;nbsp; obtained with the following pairs of primers: sense, TACGTGATGAGGAAAGAAGAG; antisense, TCCTCCACGCATCTTTACCTTG; sense, CACATTGTACGCATGGAAGAG; antisense, AGCACCTCCC AT ACGGATTC, respectively&lt;br/&gt; &lt;br/&gt; Cla12NCO-Cx43 construct template was obtained with the following pair of primers: sense, CTGCTTCGTACTTACATCATC; antisense, GAACAC GTGCGCCAGGTAC&lt;br/&gt; &lt;br/&gt; ILs of Cx50 (residues 98&amp;ndash;148) or Cx46 (residues 98&amp;ndash;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&amp;rsquo;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&amp;amp;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&amp;nbsp; 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&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>111-295-003</td><td>For anti-AQP0&amp;nbsp; 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。

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

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 

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。

重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中

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-rcas-retrovirus-into-embryonic-chicken

Research

JoVE Journal

Biology

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

905 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 

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

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

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

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

在小鸡视网膜有效的基因转移的原代细胞培养研究:<em&gt;前OVO</em&gt;电穿孔法

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

科研

发育生物学

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

癌症研究

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

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

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

鸡胚全视网膜外植体电穿孔和化学试剂处理

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

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

医学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

神经科学

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

在 奥沃 鸡胚血管内注射

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

在OVO在鸡胚视网膜电穿孔

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

生物学

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中

副本

摘要

探索更多视频

此视频中的章节

在小鸡视网膜有效的基因转移的原代细胞培养研究:

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

鸡胚全视网膜外植体电穿孔和化学试剂处理

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

基于微注射的免疫胚胎心肌细胞体内植入系统

在奥沃鸡胚血管内注射

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

在OVO在鸡胚视网膜电穿孔

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中

副本

摘要

探索更多视频

此视频中的章节

在小鸡视网膜有效的基因转移的原代细胞培养研究:

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

鸡胚全视网膜外植体电穿孔和化学试剂处理

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

基于微注射的免疫胚胎心肌细胞体内植入系统

在 奥沃 鸡胚血管内注射

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

在OVO在鸡胚视网膜电穿孔

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

在奥沃鸡胚血管内注射