Name: rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus
Uploaded: 2011-10-16T14:00:00
Description: Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

作者非常感谢协助与显微注射，以及匿名审稿的有见地的意见RJ索默和X王。这项工作是支持由NIH资助SC2GM089602。

1。转基因：DNA的制备<ol><li>占主导地位的合作注入标记：pRL3 [PPA - PRL - 1（ tu92）] pRL3质粒是一个占主导地位的合作注入的视觉识别的成功转型事件的标记。这种质粒编码为主导的PPA - PRL - 1基因密切相关的SQT - 1 C.胶原蛋白基因的突变等位基因 （ tu92） 线虫和野生型正弦运动转换成沿顺时针方向扭蠕虫的体轴1,5的议案。转化的动物是非?...

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	<li>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> Wormbook. , (2006).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.+elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li><li>Junio, A. J., Li, X., Massery, H. C., Nolan, T. J., Lamitina, S. T., Sundaram, M. V., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis:+cell-+and+tissue-specific+transgene+expression+and+co-transformation+with+vector+constructs+incorporating+a+common+multifunctional+3'+UTR.">Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR.</a> Exp. Parasitology. 118, 253-265 (2008).</li><li>Schlager, B., Wang, X., Braach, G., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+dominant+roller+mutant+and+establishment+of+DNA-mediated+transformation+in+the+nematode+Pristionchus+pacificus.">Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus.</a> Genesis. 47, 300-304 (2009).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pristionchus+pacificus:+a+well-rounded+nematode.">Pristionchus pacificus: a well-rounded nematode.</a> Bioessays. 28, 651-659 (2006).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattraction+in+Pristionchus+nematodes+and+implications+for+insect+recognition.">Chemoattraction in Pristionchus nematodes and implications for insect recognition.</a> Curr. Biol. 16, 2359-2365 (2006).</li><li>Rudel, D., Riebesell, M., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadogenesis+in+Pristionchus+pacificus+and+organ+evolution:+development,+adult+morphology+and+cell-cell+interactions+in+the+hermaphrodite+gonad.">Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad.</a> Dev. Biol. 277, 200-221 (2005).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 11, 802-805 (1994).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Worm+Lab:+from+Culturing+Worms+to+Mutagenesis.">An Introduction to Worm Lab: from Culturing Worms to Mutagenesis.</a> J. Vis. Exp. (47), e2293-e2293 (2011).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+Microinjection.">Transformation and Microinjection.</a> Wormbook. , (2006).</li></ol>

P. pacificus人群中找到全球各种金龟子甲虫物种的密切联系，是一个模型之间的自由生活线虫和寄生线虫中间。 体育的力量作为一个新兴的模式生物pacificus在其遗传和物理图谱，促进不偏不倚正向遗传筛选分离突变体的位置映射整合打下（即不只是以前线虫为特征的候选基因）6,10。然而，C线虫基因技术是不会轻易转让给体育pacificus由于显著?...

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rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus

虽然它是，越来越多的新兴模式生物实惠，进一步深入的基因功能和表达分析RNA干扰和稳定的转基因获得完全基因组测序仍然在许多物种由于特定的解剖和分子细胞生物学的有机体有限。例如，外冠集团线虫线虫 3，稳定传输的非线虫品种的转基因株系，其中包括没有在这似是而非的门（线虫）报道，除粪类 圆 线虫4 Pristionchus pacificus 5。为了方便的P.作用不断扩大pacificus的发展，演变和行为6-7的研究，我们在这里描述当前用于显微注射转基因动物和基因敲除通过RNAi的方法。性腺一样<em&gt; C。线虫和其他大多数线虫，P 的性腺pacificus合胞和能够纳入直接显微注射交付时的卵母细胞的DNA和RNA。不像C。线虫然而，稳定的转基因继承和躯体表达在体育pacificus需要的基因组与目标的转基因和共注射标记 5的两端互补的核酸内切酶酶切的DNA自我此外。此外载体的基因组DNA是类似粪类圆线虫 4和C 的生殖细胞转基因表达的要求线虫 。但是，目前尚不清楚如果自己的基因组DNA为动物的具体要求是因为体育pacificus SOMA是非常有效的，在沉默不复杂的多拷贝基因或染色体外体育阵列pacificus需要在有丝分裂过程中适当的动粒组装的基因组序列。两个武装（腹侧迁移didelphic）雌雄同体性腺进一步复杂化的能力， 在 8个人蠕虫都注入生殖腺。我们也证明了利用显微注射，以击倒一个显性突变（滚子，tu92）由双链RNA（dsRNA）的注入性腺获得非滚动F 1子代。不像C。线虫 ，但最喜欢的其他线虫， 体育pacificus PS312是不能接受系统性的RNAi通过喂养和浸泡，因此双链RNA必须注射到合胞性腺管理。在当前的研究中，我们希望描述的显微注射过程中， 需要变换PPA - EGL - 4启动子：：GFP融合的记者和击倒主导辊PRL - 1（tu92）突变的视觉信息的协议。

Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

在模式生物，转基因操纵基因的功能，而RNAi能够敲除特定的mRNA转录1-2。该协议的目的是为了说明需要稳定传输的DNA和瞬态双链RNA引入necromenic线虫技术<em&gt; Pristionchus pacificus</em在进化，发育和行为生物学的研究。

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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Biology

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

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科研

生物学

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Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

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慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...