Name: loss-of-function approach in the embryonic chick retina by using tol2 transposon-mediated transgenic expression of artificial micrornas
Uploaded: 2022-05-18T14:00:00
Description: Watch this Scientific Journal Video about Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs at JoVE.com

pT2K-CAGGS和pCAGGS-T2TP载体分别由Yoshiko Takahashi（日本京都京都大学）和Koichi Kawakami（日本三岛国立遗传学研究所）提供。我们感谢Michael Berberoglu对手稿的重要阅读。这项工作得到了皇家学会和生物技术与生物科学研究委员会（BBSRC）（英国）对M.N.的资助。

1. miRNA表达载体的构建
注意：构建miRNA表达载体的程序（步骤1.1-1.3,1.5-1.6）针对miRNA表达试剂盒进行了优化，即带有EmGFP的Block-iT Pol II miR RNA表达试剂盒，如前所述15,16。该试剂盒提供了旨在允许miRNA表达的表达载体（pcDNA6.2-GW/EmGFP-miRNA）、对照载体（pcDNA6.2-GW/EmGFP-miRNA阴性对照质粒）、辅助试剂和生产miRNA表达载体的说明（?...

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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=RCAS-RNAi:+a+loss-of-function+method+for+the+developing+chick+retina.">RCAS-RNAi: a loss-of-function method for the developing chick retina.</a> BMC Developmental Biology. 6, 2 (2006).</li><li>Nakamura, H., Funahashi, J. <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+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Koga, A., Iida, A., Hori, H., Shimada, A., Shima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+DNA+transposon+as+a+natural+mutator:+the+medaka+fish+Tol2+element+contributes+to+genetic+variation+without+recognizable+traces.">Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces.</a> Molecular Biology and Evolution. 23 (7), 1414-1419 (2006).</li><li>Kawakami, K., Shima, A., Kawakami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+functional+transposase+of+the+Tol2+element,+an+Ac-like+element+from+the+Japanese+medaka+fish,+and+its+transposition+in+the+zebrafish+germ+lineage.">Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (21), 11403-11408 (2000).</li><li>Kawakami, K., 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=A+transposon-mediated+gene+trap+approach+identifies+developmentally+regulated+genes+in+zebrafish.">A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.</a> Developmental Cell. 7 (1), 133-144 (2004).</li><li>Kawakami, K., Imanaka, K., Itoh, M., Taira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excision+of+the+Tol2+transposable+element+of+the+medaka+fish+Oryzias+latipes+in+Xenopus+laevis+and+Xenopus+tropicalis.">Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis.</a> Gene. 338 (1), 93-98 (2004).</li><li>Sato, 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=Stable+integration+and+conditional+expression+of+electroporated+transgenes+in+chicken+embryos.">Stable integration and conditional expression of electroporated transgenes in chicken embryos.</a> Developmental Biology. 2 (2), 616-624 (2007).</li><li>Kawakami, K., Noda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+the+Tol2+element,+an+Ac-like+element+from+the+Japanese+medaka+fish+Oryzias+latipes,+in+mouse+embryonic+stem+cells.">Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells.</a> Genetics. 166 (2), 895-899 (2004).</li><li>Hou, X., 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=Conditional+knockdown+of+target+gene+expression+by+tetracycline+regulated+transcription+of+double+strand+RNA.">Conditional knockdown of target gene expression by tetracycline regulated transcription of double strand RNA.</a> Development, Growth &amp; Differentiation. 53, 69-75 (2011).</li><li>Nakamoto, C., 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=Nel+positively+regulates+the+genesis+of+retinal+ganglion+cells+by+promoting+their+differentiation+and+survival+during+development.">Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development.</a> Molecular Biology of the Cell. 25 (2), 234-244 (2014).</li><li>Nakamoto, M., Nakamoto, C., Mao, C. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> iRetinal Development: Methods and Protocols. Vol. 2092 Methods in Molecular Biology. 8, 91-108 (2020).</li><li>BLOCK-iT PolII miR RNAi Expression Vector Kits, User Manual Pol II miR RNAi Expression Vector Kits. Invitrogen Available from: <a target="_blank" href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets/LSG/manuals/blockit_miRNAexpressionvector_man.pdf&amp;title=BLOCK-iT&amp;trade">https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets/LSG/manuals/blockit_miRNAexpressionvector_man.pdf&amp;title=BLOCK-iT&amp;trade</a> (2021)</li><li>Flanagan, J. G., 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=Alkaline+phosphatase+fusions+of+ligands+or+receptors+as+in+situ+probes+for+staining+of+cells,+tissues,+and+embryos.">Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues, and embryos.</a> Methods in Enzymology. 327, 19-35 (2000).</li><li>Hamburger, V., Hamilton, H. I. <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, 49-92 (1951).</li><li>Matsuhashi, 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=New+gene,+nel,+encoding+a+M(r)+93+K+protein+with+EGF-like+repeats+is+strongly+expressed+in+neural+tissues+of+early+stage+chick+embryos.">New gene, nel, encoding a M(r) 93 K protein with EGF-like repeats is strongly expressed in neural tissues of early stage chick embryos.</a> Developmental Dynamics. 203 (2), 212-222 (1995).</li><li>Matsuhashi, 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=New+gene,+nel,+encoding+a+Mr+91+K+protein+with+EGF-like+repeats+is+strongly+expressed+in+neural+tissues+of+early+stage+chick+embryos.">New gene, nel, encoding a Mr 91 K protein with EGF-like repeats is strongly expressed in neural tissues of early stage chick embryos.</a> Developmental Dynamics. 207 (2), 233-234 (1996).</li><li>Jiang, 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=In+vitro+guidance+of+retinal+axons+by+a+tectal+lamina-specific+glycoprotein+Nel.">In vitro guidance of retinal axons by a tectal lamina-specific glycoprotein Nel.</a> Molecular and Cellular Neuroscience. 41 (2), 113-119 (2009).</li><li>Nakamura, R., Nakamoto, C., Obama, H., Durward, E., Nakamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+analysis+of+Nel,+a+Thrombospondin-1-like+glycoprotein+involved+in+neural+development+and+functions.">Structure-function analysis of Nel, a Thrombospondin-1-like glycoprotein involved in neural development and functions.</a> Journal of Biological Chemistry. 287 (5), 3282-3291 (2012).</li><li>Nakamoto, C., Durward, E., Horie, M., Nakamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nell2+regulates+the+contralateral-versus-ipsilateral+visual+projection+as+a+domain-specific+positional+cue.">Nell2 regulates the contralateral-versus-ipsilateral visual projection as a domain-specific positional cue.</a> Development. 146 (4), (2019).</li><li>Yee, J. 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该协议提供了通过使用 卵 电穿孔和 Tol2 转座子系统中人工 miRNA 的转基因表达在发育中的鸡视网膜中基因沉默的详细指南。
以下因素对于成功执行此技术至关重要。首先，使用经证实具有强大敲低效果的miRNA序列至关重要。在将它们应用于 卵 电穿孔之前，在 体外 测定中测试单个前 miRNA 序列的基因抑制效率（步骤 1.4），并链上显示最高敲低活性的两个前...

脊椎动物视网膜是研究神经发育的重要模型系统。尽管视网膜位于外围位置，但在解剖学和发育上是中枢神经系统的延伸，视神经由视网膜神经节细胞的轴突组成，代表中枢神经系统内的束。小鸡视网膜作为研究神经发育分子机制的模型系统具有显著优势：体型大，发育迅速;它与人类视网膜具有结构和功能相似性;它很容易用于可视化和实验操作。利用鸡视网膜广泛研究了神经发育过程中细胞增殖和分化、形态发生和轴突引导的分子机制。
在过去的二十年中，卵子电穿孔已成功用于将异位基因引入发育中的雏鸡胚胎中的细胞中。该技术允许标记发育中的细胞，细胞命运追踪，细胞迁移和轴突束的追踪，以及异位基因表达，用于基因功能的体内分析。卵内电穿孔在鸡胚胎中有效表达异位基因的条件已经确立1,2,3。
尽管有这些优点，但缺乏用于基因功能研究的稳定功能丧失技术一直是雏鸡胚胎的主要技术限制。虽然用小干扰RNA（siRNA）4或短发夹RNA（shRNA）5的表达载体电穿孔的鸡胚胎显示出靶基因的敲低，但这些方法中的基因抑制是短暂的，因为一旦细胞失去引入的RNA或DNA，这种影响就会消失。通过RCAS（R转换Competent A型肉瘤-白血病病毒（ASLV）长末端重复（LTR）与S板受体）逆转录病毒系统将siRNA递送到雏鸡胚胎中可以实现更稳定的基因抑制6。病毒载体整合到宿主基因组中，异位基因稳定表达。然而，逆转录病毒只能在细胞周期的有丝分裂（M）阶段整合到分裂细胞的基因组中，这可能会对可以应用这种功能丧失方法的发育阶段和/或细胞类型施加限制。此外，RCAS对转基因的表达似乎比卵电穿孔诱导的表达更慢且更不稳定7。
转座子是从基因组上的一个位置移动到另一个位置的遗传元件。Tol2元件是hAT转座元件家族的成员，包含一个编码转座酶的内部基因，该转座酶催化Tol2元件8的转座子反应。当携带基因表达盒的质粒载体被引入具有Tol2转座酶表达构建体的脊椎动物细胞中时，将携带基因表达盒的质粒载体，两侧是Tol2元件的左端和右端序列（分别为200 bp和150 bp），将表达盒从质粒中切除并整合到宿主基因组中，从而支持异位基因的稳定表达（图1).已经表明，Tol2转座元件可以非常有效地诱导不同脊椎动物物种的基因转位，包括斑马鱼9,10，青蛙11，小鸡12和小鼠13，因此是一种有用的转基因和插入诱变方法。Tol2转座子系统已成功用于通过从长双链RNA14加工的siRNA的基因组整合来条件敲低靶基因。
该协议描述了雏鸡胚胎中的功能丧失方法，该方法涉及通过Tol2转座子系统引入人工microRNA（miRNA）15,16。在这种方法中，将EmGFP（翡翠绿色荧光蛋白）标记物的表达盒和针对靶基因的人工miRNA克隆到Tol2转座子载体中。然后将Tol2转座子构建体通过卵电穿孔将Tol2转座酶表达构建体引入胚胎小鸡视网膜中。在转染的视网膜细胞中，转座酶催化从转座子载体中切除表达盒并将其整合到宿主染色体中，从而导致miRNA和EmGFP蛋白的稳定表达。在我们之前的研究中，我们成功地敲低了Nel的表达，Nel是一种主要在神经系统中表达的细胞外糖蛋白，在发育中的小鸡视网膜中（见代表性结果）。我们的结果表明，通过该技术可以在卵中实现稳定有效的基因抑制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G needle, 2&#34;</td>
			<td>VWR</td>
			<td>89219-320</td>
			<td></td>
		</tr>
		<tr>
			<td>AP-TAG kit A and AP-TAG kit B</td>
			<td>GenHunter Corp</td>
			<td>Q201 and Q202</td>
			<td>Plasmid vectors for making AP fusion proteins (https://www.genhunter.com/products/ap-tag-kit-a.html, https://www.genhunter.com/products/ap-tag-kit-b.html)</td>
		</tr>
		<tr>
			<td>Block-iT RNAi Designer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>An online tool to choose target sequences and design pre-miRNA sequences (https://rnaidesigner.thermofisher.com/rnaiexpress/)</td>
		</tr>
		<tr>
			<td>BSA 10 mg</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>C115CB cables</td>
			<td>Sonidel</td>
			<td>C115CB</td>
			<td>https://www.sonidel.com/product_info.php?products_id&#188;254</td>
		</tr>
		<tr>
			<td>C117 cables</td>
			<td>Sonidel</td>
			<td>C117</td>
			<td>https://www.sonidel.com/product_info.php?products_id&#188;252</td>
		</tr>
		<tr>
			<td>Capillary tubes with omega dot fiber (Micropipette needles)</td>
			<td>FHC</td>
			<td>30-30-1</td>
			<td>1 mm O.D. 0.75 mm I.D</td>
		</tr>
		<tr>
			<td>CUY21 square wave electroporator</td>
			<td>Nepa Gene</td>
			<td>CUY21</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethanolamine (pH 9.8)</td>
			<td>Sigma-Aldrich</td>
			<td>D8885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Kurl</td>
			<td>B-Lab-600-110</td>
			<td>https://www.flemingoutdoors.com/kuhl%2D%2D-600-egglaboratory-incubator%2D%2D-b-lab-600-110.html</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td>Sonidel</td>
			<td>CUY580</td>
			<td>https://www.sonidel.com/product_info.php?products_id&#188;85</td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td>Nepa Gene</td>
			<td>CUY611P3-1</td>
			<td>https://www.sonidel.com/product_info.php?products_id&#188;94</td>
		</tr>
		<tr>
			<td>Electromax DH10B</td>
			<td>Invitrogen</td>
			<td>18290-015</td>
			<td>Electrocompetent E. coli cells</td>
		</tr>
		<tr>
			<td>Fast green FCF</td>
			<td>Sigma-Aldrich</td>
			<td>F7258</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertilized chicken eggs (Gallus gallus)</td>
			<td>Obtained from commercial vendors (e.g. Charles River) or local farmers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gooseneck fiber light source</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FuGene 6 transfection reagent</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (50 &#956;L)</td>
			<td>Sigma-Aldrich</td>
			<td>20715</td>
			<td>Hamilton Cat No&#160; 80901</td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt solution</td>
			<td>Sigma-Aldrich</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Heavy mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>330760</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>GIBCO</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Homoarginine</td>
			<td>Sigma-Aldrich</td>
			<td>H10007</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>13112</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige (Japan)</td>
			<td>MM3</td>
			<td>http://products.narishige-group.com/group1/MM-3/electro/english.html</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Shutter Instrument</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Nitrophenylphosphate</td>
			<td>Sigma-Aldrich</td>
			<td>20-106</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAGGS-T2TP vector</td>
			<td></td>
			<td></td>
			<td>Tol2 transposase expression plasmid. A generous kind gift of Koichi Kawakami (National Institute of Genetics, Japan). Also available from Addgene.</td>
		</tr>
		<tr>
			<td>Pfu</td>
			<td>ThermoFisher</td>
			<td>F566S</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer (Optional)</td>
			<td>Parker</td>
			<td></td>
			<td>Pressure microinjection system</td>
		</tr>
		<tr>
			<td>Plasmid maxi kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Plasmid maxiprep kit</td>
		</tr>
		<tr>
			<td>pT2K-CAGGS vector</td>
			<td></td>
			<td></td>
			<td>Tol2 transposon vector. Kindly provided by Yoshiko Takahashi (Kyoto University, Japan)</td>
		</tr>
		<tr>
			<td>PVC tubing</td>
			<td>VWR (UK)</td>
			<td>228-3830</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S9007-5</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Promega</td>
			<td>M1801</td>
			<td></td>
		</tr>
		<tr>
			<td>The BLOCK-iT Pol II miR RNA expression kit with EmGFP</td>
			<td>Invitrogen</td>
			<td>K493600</td>
			<td>Contains the miRNA expression vector (pcDNA6.2-GW/EmGFP-miRNA), a control vector (pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid), accessory reagents, and instructions (https://www.thermofisher.com/order/catalog/product/K493600?SID.srch-hj-K4936-00)</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

loss-of-function approach in the embryonic chick retina by using tol2 transposon-mediated transgenic expression of artificial micrornas

长期以来，小鸡视网膜一直是发育神经生物学中重要的模型系统，其优点包括体积大、发育迅速以及可视化和实验操作的可访问性。然而，其主要技术局限性是缺乏用于基因功能分析的强有力的功能丧失方法。该协议描述了一种在发育中的鸡视网膜中进行基因沉默的方法，该方法涉及通过使用Tol2转座子系统进行人工microRNA（miRNA）的转基因表达。在这种方法中，将包含EmGFP（翡翠绿色荧光蛋白）标记物表达盒和针对靶基因的人工前miRNA序列的Tol2转座子质粒引入胚胎小鸡视网膜中，并通过 卵内 电穿孔构建Tol2转座酶表达。在转染的视网膜细胞中，转座酶催化从转座子载体中切除表达盒并将其整合到宿主染色体中，从而导致miRNA和EmGFP蛋白的稳定表达。在我们之前的研究中，我们已经证明，Nel（一种在神经发育中发挥多种功能的糖蛋白）的表达可以通过使用该技术在发育中的小鸡视网膜中显着抑制。我们的结果表明，这种方法诱导了对基因表达的稳定和强大的抑制，从而为视网膜发育的研究提供了一种有效的功能丧失方法。

构建Tol2转座子构建体，用于表达针对Nel的人工miRNA Nel（Neural Epidermal Growth Factor （EGF）-Like;也称为Nell2）是一种细胞外糖蛋白。它与血小板反应素-1具有结构相似性，主要在神经系统中表达20,21。我们之前已经证明Nel调节视网膜神经节细胞的分化和存活15，并作为视网膜轴突22,23,24的抑制引导线索。<sup class="...

Watch this Scientific Journal Video about Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs at JoVE.com

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

我们开发了一种新颖的功能丧失方法，该方法涉及通过使用 卵 电穿孔和Tol2转座子系统将人工微RNA序列引入和基因组整合到鸡胚胎中。该技术为发育过程中的基因功能研究提供了一种强大而稳定的基因敲低方法。

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

该方法为研究雏鸡视网膜发育提供了一种功能丧失方法。该技术支持快速和持续地抑制视网膜特定区域中的靶基因。这种方法也可以应用于雏鸡胚胎的其他部分，包括神经和非神经组织。对于卵电穿孔，通过将25毫克的Fast Green FCF溶解在10毫升PBS中来制备0.25%的Fast Green Solution，然后使用0.2微米的注射器过滤器过滤溶液。接下来，通过将microRNA祖母绿GFP质粒与Tol2转座酶表达质粒以2：1的比例混合来制备DNA鸡尾酒注射溶液。然后将制备的Fast Green溶液以1：10的比例添加到DNA混合物中，以可视化注射区域。接下来，设置显微注射装置。该设备由汉密尔顿注射器、18 号两英寸针、两厘米长的聚氯乙烯管和微量移液器针组成。用重矿物油填充汉密尔顿注射器，然后将18号针头连接到注射器上，并通过按下注射器柱塞来填充针头的内部空间。接下来，将聚氯乙烯管连接到针头的末端，并在管子中填充油。将拉动的微量移液器针头连接到管子上。然后在使用细镊子的解剖显微镜下，将针尖折断至直径为10至20微米，以开一个小口。用油填充整个针头。接下来，将五微升彩色DNA鸡尾酒放入培养皿上。在解剖显微镜下，将微量移液器针头的尖端放入培养皿上的DNA溶液中，然后将溶液缓慢抽入针中。在针头内外的压力平衡后，将针尖从DNA溶液中取出，并将其浸没在无菌PBS中的小烧杯中直至注射。然后要设置电穿孔设备，请将一对铂电极牢固地固定在显微操纵器上。将电极尖端之间的间距调整为两毫米，并使用电缆将电极连接到方波脉冲发生器。接下来，对于DNA显微注射，从培养箱中取出受精白里格角牛卵，并用浸泡在70%乙醇中的薄纸擦拭鸡蛋表面。然后将 18 号针头连接到 10 毫升注射器上，并将针头插入鸡蛋的钝端，向下倾斜以避免损坏蛋黄。从鸡蛋中取出两到三毫升白蛋白后，用一块透明胶带密封孔，并用光烛蜡烛，以确保胚胎和卵黄膜与壳分离。接下来，使用镊子从鸡蛋顶部取出一块蛋壳，露出胚胎。任何时候不要窗口超过五个鸡蛋，以防止在电穿孔过程中胚胎变干。将针尖从其近端以45度角插入视囊泡，并通过缓慢按下柱塞直到蓝色溶液充满管腔来注射DNA混合物。取出针头，并将其尖端放回PBS。对于电穿孔，将脉冲电压设置为 15 伏，脉冲长度设置为 15 毫秒，脉冲间隔设置为 915 毫秒，脉冲数设置为 5。在胚胎上方的玻璃膜上加入几滴HBSS后，使用显微操纵器将电极降低到垂直于胚胎前后轴的HBSS中。将电极放在视囊泡的两侧，确保电极不接触胚胎或血管，然后施加脉冲电场。电穿孔完成后，取出电极，并用水浸泡的无菌棉签轻轻清洁电极，以避免白蛋白积聚。用透明胶带密封窗口，并重新孵育胚胎直到所需的发育阶段。将单个前microRNA序列转染到表达HEK293T细胞的Nel AP细胞中，导致针对核苷酸482至502、910至930和2461至2481的构建体显着抑制Nel表达。链上两个microRNA序列显著提高了敲低效率。与未链的单个前microRNA序列相比，所有三种组合均显示出增强的抑制活性。在转染后至少13天观察到强烈的抑制。当靶向核苷酸 482 至 502 和 2461 至 2481 的 Nel 前 microRNA 构建体与转座酶表达载体共转染到鸡视网膜中时，在胚胎第 4.5 天，表达祖母绿 GFP 的细胞中视网膜色素上皮中的 Nel 表达显着降低。在八天大的胚胎中，Nel在视网膜神经节细胞中的表达也下降。相比之下，视网膜中的Nel表达不受对照microRNA引入的影响。按照该程序，可以使用各种方法检查对靶基因抑制的影响，例如检查细胞增殖和分化，细胞死亡以及细胞和轴突示踪。

Research

JoVE Journal

Neuroscience

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

科研

神经科学

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

In ovo Expression of MicroRNA in Ventral Chick Midbrain

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...