Development of a microinjection system for RNA interference in the water flea Daphnia pulex.
Development of a microinjection system for RNA interference in the water flea Daphnia pulex.
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DOI：
10.1186/1472-6750-13-96
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发表时间：
2013-11-05
影响因子：
3.5
通讯作者：
Iguchi T
中科院分区：
文献类型：
--
作者：
Hiruta C;Toyota K;Miyakawa H;Ogino Y;Miyagawa S;Tatarazako N;Shaw JR;Iguchi T
普遍存在的淡水微型甲壳动物蚤状水蚤（Daphnia pulex）为人类健康研究和监测生态系统完整性提供了模型系统。它是第一个有很好注释的甲壳类动物，参考基因组组装揭示了一个异常高的基因计数，突出了一个大的基因孤儿院，即，以前没有的基因。水蚤能够进行无性繁殖或有性繁殖，这使得它们非常适合进行基因操作，但需要建立基因操作技术来准确定义基因功能。虽然以前的研究开发了一个RNA干扰（RNAi）系统的一个同源D。magna，这些方法不适用于D.因为它们早期的胚胎体积较小。在这些研究中，我们开发了针对D.通过首先确定其分离胚胎的最佳培养条件，然后将这些条件应用于显微注射技术和原理验证RNAi实验的发展，来研究蚤状蚤。我们发现，分离的胚胎最好培养在2%的琼脂板上，浸泡在60 mM蔗糖溶解在M4培养基，提供最佳条件显微注射。然后，我们注射了特异于Distal-less基因（Dll）的双链（ds）RNA，Dll是无脊椎动物和脊椎动物肢体发育所必需的同源框转录因子。注射的胚胎在第二触角和附属物发育中呈现缺陷，并且dsRNA诱导Dll mRNA的降解，表明该技术成功地抑制了靶基因的转录。我们建立了一套用于D.蚤。这些技术增加了不断增长的基因组工具箱，并提高了环境，进化和发育基因组学这一重要模型的遗传易处理性。
The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna, these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments. We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene. We developed a microinjection system for RNAi studies in D. pulex. These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.
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DOI：
10.1073/pnas.0903105106
发表时间：
2009-08-18
影响因子：
11.1
作者：
Liubicich, Danielle M.;Serano, Julia M.;Patel, Nipam H.
通讯作者：
Patel, Nipam H.
DOI：
10.1126/science.1197761
发表时间：
2011-02-04
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Colbourne JK;Pfrender ME;Gilbert D;Thomas WK;Tucker A;Oakley TH;Tokishita S;Aerts A;Arnold GJ;Basu MK;Bauer DJ;Cáceres CE;Carmel L;Casola C;Choi JH;Detter JC;Dong Q;Dusheyko S;Eads BD;Fröhlich T;Geiler-Samerotte KA;Gerlach D;Hatcher P;Jogdeo S;Krijgsveld J;Kriventseva EV;Kültz D;Laforsch C;Lindquist E;Lopez J;Manak JR;Muller J;Pangilinan J;Patwardhan RP;Pitluck S;Pritham EJ;Rechtsteiner A;Rho M;Rogozin IB;Sakarya O;Salamov A;Schaack S;Shapiro H;Shiga Y;Skalitzky C;Smith Z;Souvorov A;Sung W;Tang Z;Tsuchiya D;Tu H;Vos H;Wang M;Wolf YI;Yamagata H;Yamada T;Ye Y;Shaw JR;Andrews J;Crease TJ;Tang H;Lucas SM;Robertson HM;Bork P;Koonin EV;Zdobnov EM;Grigoriev IV;Lynch M;Boore JL
通讯作者：
Boore JL
影响因子：
2.7
作者：
Lohmann, JU;Endl, I;Bosch, TCG
通讯作者：
Bosch, TCG
影响因子：
12.8
作者：
ELENDT, BP;BIAS, WR
通讯作者：
BIAS, WR
影响因子：
2.4
作者：
Schoppmeier, M;Damen, WGM
通讯作者：
Damen, WGM