Name: use of a recombinant mosquito densovirus as a gene delivery vector for the functional analysis of genes in mosquito larvae
Uploaded: 2017-10-06T13:00:00
Description: Watch this Scientific Journal Video about Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae at JoVE.com

作者承认中国国家重点研究开发计划 (2016YFC1200500)、中国国家自然科学基金 (81672054 和 81371846)、自然研究团队计划的资助。广东省科学基金 (2014A030312016) 和广州科技计划 (201508020263)。我们感激地承认教授乔纳森 (科罗拉多州立大学) 提供 pUCA 和 p7NS1-GFP 质粒和批判阅读这篇手稿。

所有协议均由南方医科大学伦理委员会批准. 
 1. 设计人工内含子 注意: 在这项工作中使用的人工内含子长度为 65 bp, 序列为5和 #39;-GTAAGAGTCGATCGACGCGTTACTAACTGGTACCTCTTCTTTTTTTTTTGATATCCTGCAG-3 和 #39;. 
<ol> <li> 将 hpa i 限制站点放在人工内含子的两端, 使 hpa i 消化可以从质粒中释放内含子 (见 图 1 A ). </li>
	<li> 将?...

<ol>
	<li>Tolle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito-borne+diseases.">Mosquito-borne diseases.</a> Current problems in pediatric and adolescent health care. 39, 97-140 (2009).</li><li>Petersen, L. R., Jamieson, D. J., Powers, A. M., Honein, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus.">Zika Virus.</a> The New England journal of medicine. 374, 1552-1563 (2016).</li><li>Roberts, D. R., Andre, 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=Insecticide+resistance+issues+in+vector-borne+disease+control.">Insecticide resistance issues in vector-borne disease control.</a> The American journal of tropical medicine and hygiene. 50, 21-34 (1994).</li><li>Attaran, A., Roberts, D. R., Curtis, C. F., Kilama, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+risks+on+the+backs+of+the+poor.">Balancing risks on the backs of the poor.</a> Nature medicine. 6, 729-731 (2000).</li><li>Volohonsky, 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=Transgenic+Expression+of+the+Anti-parasitic+Factor+TEP1+in+the+Malaria+Mosquito+Anopheles+gambiae.">Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae.</a> PLoS pathogens. 13, 1006113 (2017).</li><li>Galizi, R., 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+synthetic+sex+ratio+distortion+system+for+the+control+of+the+human+malaria+mosquito.">A synthetic sex ratio distortion system for the control of the human malaria mosquito.</a> Nature communications. 5, 3977 (2014).</li><li>Bourtzis, K., Lees, R. S., Hendrichs, J., Vreysen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+one+rabbit+out+of+the+hat:+Radiation,+transgenic+and+symbiont-based+approaches+for+sustainable+management+of+mosquito+and+tsetse+fly+populations.">More than one rabbit out of the hat: Radiation, transgenic and symbiont-based approaches for sustainable management of mosquito and tsetse fly populations.</a> Acta tropica. 157, 115-130 (2016).</li><li>Kumar, S. S., Puttaraju, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvised+microinjection+technique+for+mosquito+vectors.">Improvised microinjection technique for mosquito vectors.</a> The Indian journal of medical research. 136, 971-978 (2012).</li><li>Attardo, G. M., Higgs, S., Klingler, K. A., Vanlandingham, D. L., Raikhel, 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=RNA+interference-mediated+knockdown+of+a+GATA+factor+reveals+a+link+to+anautogeny+in+the+mosquito+Aedes+aegypti.">RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito Aedes aegypti.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 13374-13379 (2003).</li><li>Singh, A. D., Wong, S., Ryan, C. P., Whyard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+delivery+of+double-stranded+RNA+in+larvae+of+the+yellow+fever+mosquito,+Aedes+aegypti:+implications+for+pest+mosquito+control.">Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: implications for pest mosquito control.</a> Journal of insect science. 13, 69 (2013).</li><li>Ward, T. W., 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=Aedes+aegypti+transducing+densovirus+pathogenesis+and+expression+in+Aedes+aegypti+and+Anopheles+gambiae+larvae.">Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae.</a> Insect molecular biology. 10, 397-405 (2001).</li><li>Carlson, J., Suchman, E., Buchatsky, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Densoviruses+for+control+and+genetic+manipulation+of+mosquitoes.">Densoviruses for control and genetic manipulation of mosquitoes.</a> Advances in virus research. 68, 361-392 (2006).</li><li>Barreau, C., Jousset, F. X., Bergoin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+of+the+Aedes+albopictus+parvovirus+(AaPV),+a+denso-like+virus,+for+Aedes+aegypti+mosquitoes.">Pathogenicity of the Aedes albopictus parvovirus (AaPV), a denso-like virus, for Aedes aegypti mosquitoes.</a> Journal of invertebrate pathology. 68, 299-309 (1996).</li><li>Liu, P., 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=Development+of+non-defective+recombinant+densovirus+vectors+for+microRNA+delivery+in+the+invasive+vector+mosquito,+Aedes+albopictus.">Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.</a> Scientific reports. 6, 20979 (2016).</li><li>Ortega, M. M., Bouamar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+Designing+MicroRNA+Sponges:+From+Construction+to+Stable+Cell+Line.">Guidelines on Designing MicroRNA Sponges: From Construction to Stable Cell Line.</a> Methods in molecular biology. 1509, 221-233 (2017).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli+using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli using the heat shock method.</a> Journal of visualized experiments : JoVE. , e253 (2007).</li><li>Afanasiev, B. N., Kozlov, Y. V., Carlson, J. O., Beaty, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Densovirus+of+Aedes+aegypti+as+an+expression+vector+in+mosquito+cells.">Densovirus of Aedes aegypti as an expression vector in mosquito cells.</a> Experimental parasitology. 79, 322-339 (1994).</li><li>Hu, H. 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=Evolution+of+the+human-specific+microRNA+miR-941.">Evolution of the human-specific microRNA miR-941.</a> Nature communications. 3, 1145 (2012).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 74, 5463-5467 (1977).</li><li>Dalby, B., 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=Advanced+transfection+with+Lipofectamine+2000+reagent:+primary+neurons,+siRNA,+and+high-throughput+applications.">Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications.</a> Methods (San Diego, Calif). 33, 95-103 (2004).</li><li>Gu, J. B., Dong, Y. Q., Peng, H. J., Chen, X. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombinant+AeDNA+containing+the+insect-specific+toxin,+BmK+IT1,+displayed+an+increasing+pathogenicity+on+Aedes+albopictus.">A recombinant AeDNA containing the insect-specific toxin, BmK IT1, displayed an increasing pathogenicity on Aedes albopictus.</a> The American journal of tropical medicine and hygiene. 83, 614-623 (2010).</li><li>Hou, Y., Zhang, H., Miranda, L., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serious+overestimation+in+quantitative+PCR+by+circular+(supercoiled)+plasmid+standard:+microalgal+pcna+as+the+model+gene.">Serious overestimation in quantitative PCR by circular (supercoiled) plasmid standard: microalgal pcna as the model gene.</a> PloS one. 5, 9545 (2010).</li><li>Ding, 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=Preparation+of+rAAV9+to+Overexpress+or+Knockdown+Genes+in+Mouse+Hearts.">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts.</a> Journal of visualized experiments : JoVE. , (2016).</li></ol>

重要的是要克服限制 rAaeDV 建设的两个关键障碍。第一是生产有缺陷的重组病毒。据报道, MDV 可以作为一个载体来表达适当大小的外来基因, 如蝎虫特异 neurotoxins20 和 GFP 蛋白;然而 , 无论建设策略 , 一旦码的 MDV 入或替换为外源基因 , 病毒不能独立形成 , 除非帮助的质粒是 cotransfected 提供缺少的不可缺少的病毒蛋白。有缺陷的病毒是无法参与二次传播, 这发生在体内蚊子与缺陷的基因组。我?...

蚊子传播的疾病, 如疟疾、登革热、zika 发烧和黄热病, 是主要的国际公共卫生问题, 继续占全球传染病负担的很大一部分1,2。传统的杀虫剂, 已被用于响应的载体, 是一个主要组成部分的可持续的综合防治蚊子传播疾病的控制战略。然而, 这种战略被证明是相对无效或不可取的, 由于相关的负面环境影响和蚊子的抗药性的演变3,4。由于这些原因, 迫切需要采取替代控制蚊子的方法, 利用转基因方法生产无菌的雄性蚊子, 并释放抗病菌蚊子, 这是有希望的新的控制战略。为了开发有效的新的控制方法, 如安全有效的体内基因传递, 需要对蚊子基因功能进行综合分析。
直接微注射质粒 DNA, 双链 rna (dsRNA) 或小干扰 rna (siRNA) 是最常用的体内基因传递方法的蚊子。事实上, 转基因蚊子的生产仍然依赖于一个胚胎微注射的过程5,6,7。然而, 微注射有几个局限性。首先, 这项技术要求严格, 涉及复杂的程序。其次, 注射引起对胚胎、幼虫、蛹和成虫的物理侮辱, 直接影响到目标生物体的生存能力。第三, 由于大多数生活在水生栖息地, 并具有特有的蠕动运动, 难以固定蚊子幼虫进行微注射。第四, 1st-2nd幼虫的大小为 10-20 倍, 小于 4th和老幼虫, 前者的角质层更细腻。这些特性使得对 1st-2nd幼虫的处理与在较旧的阶段相比难以操作。相结合, 这些因素有助于减少幼虫的后存活率 (约 5%), 与成人相比8。Viral-based 的分娩系统已经开发, 以克服相关的细胞外和细胞内屏障。这些系统具有易于操作、高转导效率、长期和强健的表达水平以及在体内产生持久性效果的能力。因此, 利用病毒、慢和腺的基因传递系统已被广泛应用于 inmammalian 细胞系和模型种。辛德毕斯病毒表达系统曾用于分析成年蚊子的基因功能;然而, 除了生物安全问题, 注射仍然是病毒感染的必要过程9。虽然, 在 dsRNA 溶液中浸泡幼虫的口服已被报告为一种可行的分娩方法, 但不适用于小 RNA 功能分析10。因此, 有效的病毒传递方法仍然必须为蚊子开发。
蚊子 densoviruses (MDVs) 是Parvoviridae的Densovirinae亚科的一部分, 除了一个属于Brevidensovirus11。MDV 病毒是 non-enveloped, 由单 DNA (ssDNA) 基因组和体衣壳 (直径 20 nm) 组成。病毒基因组的大小约为 4 kb, 并在宿主细胞的细胞核内复制。MDVs 在环境中相对稳定, 对蚊子具有高特异性的狭窄寄主范围。这些病毒有可能传播和保持自然在蚊子的人口, 并可以侵入这些昆虫的几乎所有器官和组织, 包括中肠, 氏小管, 脂肪体, 肌组织, 神经元和唾液腺12。
完整的 MDV 基因组可以亚成质粒载体产生 plasmid-based 感染性克隆;当这些克隆被送进蚊子细胞, 病毒基因组从质粒载体被提取, 并且传染性的病毒微粒被生产。由于 MDV 有一个小的 ssDNA 基因组, 感染性克隆很容易构建, 病毒基因组可以很容易地操作11,13。这些特性使 MDV 成为研究蚊子生物学的有价值的试剂。然而, 由于几乎所有的病毒基因组序列对病毒增殖是必不可少的, 通过替换或插入外来基因而产生的重组病毒会导致病毒包装和/或复制能力的丧失, 从而造成障碍为 MDVs 的发展作为基因交付传染媒介。在这里, 我们报告使用人工子小 rna 表达策略, 开发一个无的 rAaeDV在体内rna 传递系统, 具有方便的质粒结构的优势和维护的功能病毒, 可以产生稳定和长期的表达在宿主细胞不需要野生型病毒。此外, 这种方法可以方便幼虫的转导。
本研究介绍了以下步骤的协议: 1) rAaeDVs 编码子小 RNA 表达盒的设计 2) 利用 C6/36 包装细胞株生产重组病毒颗粒, 3) 无细胞定量分析rAaeDV 基因组复制数字, 和 4) 感染的Ae. 伊蚊幼虫直接引入病毒进入水体的幼虫栖息地。总的来说, 这项工作表明, 特定的小 rna 或靶基因可以表达或被打倒在蚊子幼虫使用发达的 MDV 分娩系统。

<table width="901">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="width:401px;height:24px;">Centrifuge machine</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">75004260</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Centrifuge System</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:119px;">363118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">GeneJET Gel Extraction Kit</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">K0691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">E.Z.N.A. Plasmid Midi Kit</td>
			<td style="width:215px;">Omega Biotech</td>
			<td style="width:119px;">D6904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Purified Agar</td>
			<td style="width:215px;">OXOID</td>
			<td style="width:119px;">LP0028A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">FastAP Thermosensitive Alkaline Phosphatase (1 U/&#181;L)</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">EF0651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">FastDigest HpaI</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">FD1034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">FastDigest XbaI</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">FD0684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">T4 DNA Ligase (5 U/&#181;L)</td>
			<td style="width:215px;">Thermo Scientific</td>
			<td style="width:119px;">EL0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">TransStbl3 Chemically Competent Cell</td>
			<td style="width:215px;">Beijing Transgen Biotech</td>
			<td style="width:119px;">CD521-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Ampicillin&#160;</td>
			<td style="width:215px;">Beijing Tiangen Biotech</td>
			<td style="width:119px;">RT501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td style="width:215px;">Gibco&#65292; Life Technologies</td>
			<td style="width:119px;">&#160;21875109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Fetal Bovine Serum&#65288; Australia Origin&#65289;</td>
			<td style="width:215px;">Gibco&#65292; Life Technologies</td>
			<td style="width:119px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">penicillin/streptomycin</td>
			<td style="width:215px;">Gibco&#65292; Life Technologies</td>
			<td style="width:119px;">15070063</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="width:401px;height:23px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:215px;">&#160;Invitrogen&#65292;Life Technologies</td>
			<td style="width:119px;">11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">Proteinase K</td>
			<td style="width:215px;">Promega</td>
			<td>MC5008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">DNase I (RNase-free)</td>
			<td style="width:215px;">&#160;Invitrogen&#65292;Life Technologies</td>
			<td style="width:119px;">AM2222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:401px;height:21px;">SuperReal PreMix Plus (SYBR Green)</td>
			<td style="width:215px;">Beijing Tiangen Biotech</td>
			<td style="width:119px;">FP205</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a recombinant mosquito densovirus as a gene delivery vector for the functional analysis of genes in mosquito larvae

体内微注射是最常用的基因转移技术, 用于分析单个蚊子的基因功能。然而, 这种方法需要一个更严格的技术要求的操作, 并涉及复杂的程序, 特别是当用于幼虫由于其体积小, 相对薄和脆弱的角质层, 和高死亡率, 这限制了它的应用。相比之下, 基因传递的病毒载体已经被开发来克服细胞外和细胞内屏障。这些系统具有操作简单, 基因转导效率高, 基因表达的长期维持, 以及在体内产生持久性效应的能力。蚊子 densoviruses (MDVs) 是特定于蚊子的、小单的 DNA 病毒, 能有效地将外来核酸转化为蚊子细胞;然而, 替换或插入外国基因来制造重组病毒通常会导致包装和/或复制能力的丧失, 这是这些病毒作为传递媒介发展的障碍。
在此, 我们报告使用人工子小 RNA 表达策略, 开发无重组的伊蚊蚊核 (AaeDV)体内交付系统。详细的程序的建设, 包装和定量分析的 rAaeDV 载体, 并为幼虫感染描述。
本研究首次论证了开发无重组 MDV 微 RNA (miRNA) 表达系统的可行性, 从而为蚊子基因的功能分析提供了有力的工具, 为病毒 paratransgenesis 在控制蚊媒疾病中的应用。我们证明, 白纹伊蚊1st幼虫可以通过将病毒引入幼虫孳生地的水体中而容易有效地感染, 而开发的 rAaeDVs 可以用来过度或击倒特定靶基因在幼虫中的表达, 为蚊子基因的功能分析提供了工具。

rAaeDV 结构的策略 产生了一个缺陷的 rAaeDV 载体来表达蚊子幼虫中的 DsRed 基因。所产生的质粒包含一个 NS1-DsRed 融合蛋白盒与 VP 蛋白删除 (图 1a)。设计的 rAaeDV 质粒包含 miRNA、miRNA 海绵、shRNA 和 amiRNA 的表达结构 (如图 1B所示)。例如, 过度 aal-let-7 在蚊子幼虫中生成了一个 rAaeDV 向量。所产?...

Watch this Scientific Journal Video about Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae at JoVE.com

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

我们报告使用人工子小 RNA 表达策略, 开发一个无重组的伊蚊蚊核 (AaeDV)体内交付系统。详细介绍了 rAaeDV 载体的构建、包装和定量分析以及幼虫感染的具体步骤。

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

Research

JoVE Journal

Developmental Biology

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

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

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

科研

发育生物学

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

A Method for Characterizing Embryogenesis in Arabidopsis

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

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

三维高频超声对小鼠早期妊娠特征的新应用

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

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...