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

लेखकों microinjection, के रूप में के रूप में अच्छी तरह से गुमनाम समीक्षक से व्यावहारिक टिप्पणियों के साथ सहायता के लिए आरजे Sommer और एक्स वैंग बहुत आभारी हैं. इस काम NIH अनुदान SC2GM089602 द्वारा समर्थित है.

1. Transgenesis: डीएनए की तैयारी <ol><li> प्रमुख सह इंजेक्शन मार्कर: pRL3 [PPA - PRL 1 (tu92)] pRL3 प्लाज्मिड नेत्रहीन सफल परिवर्तन घटनाओं की पहचान करने के लिए एक प्रमुख सह इंजेक्शन मार्कर है. यह प्लाज्मिड PPA - PRL-1 जीन की एक प...

<ol>
	<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>

पी. pacificus आबादी विभिन्न scarab बीटल दुनिया भर में प्रजातियों के साथ निकट सहयोग में पाए जाते हैं और एक मॉडल निमेटोड मुक्त रहने और परजीवी नेमाटोड के बीच मध्यवर्ती है . पी. की ताकत एक उभरती हुई मॉडल जीव के ...

<table border="1"><tbody><tr><th> उत्पाद: </th><th> Catalog #: </th><th> प्रदायक: </th></tr><tr><td> DMI3000 इंजेक्शन खुर्दबीन </td><td> DMI3000 </td><td> Leica </td></tr><tr><td> Microinjector जोड़तोड़ (डायरेक्ट ड्राइव) </td><td> Narashige BC-3 बॉल संयुक्त </td><td> Tritech अनुसंधान </td></tr><tr><td> सुई डांड़ी </td><td> Narashige पीसी - 10 </td><td> Tritech अनुसंधान </td></tr><tr><td> ™ सभी डिजिटल मल्टी दबाव प्रणाली MicroInjector </td><td> मिंज - डी Narashige </td><td> Tritech अनुसंधान </td></tr><tr><td> GeneElute ™ स्तनधारी जीनोमिक डीएनए Miniprep किट </td><td> G1N70 </td><td> सिग्मा </td></tr><tr><td> pJet क्लोनिंग जेट किट </td><td> K1231 </td><td> Fermentas </td></tr><tr><td> GeneJET प्लाज्मिड Miniprep किट </td><td> K0502 </td><td> Fermentas </td></tr><tr><td> डीएनए स्वच्छ और Concentrator ™ </td><td> D4005 </td><td> ZYMO अनुसंधान </td></tr><tr><td> ब्लॉक आईटी ™ आरएनएआई Topo ® प्रतिलेखन किट </td><td> K3500-01 और K3650-01 </td><td> Invitrogen </td></tr><tr><td> Difco ™ अग्रवाल नोबल </td><td> DF0142-15-2 </td><td> फिशर scientifi </td></tr><tr><td> माइक्रोस्कोप कवर ग्लास (1.5 - मोटी 0.16 0.19mm; आकार: 50 x 45mm) </td><td> 12-554-F </td><td> फिशर साइंटिफिक </td></tr><tr><td> ग्लास capillaries (रेशा) </td><td> 615000 </td><td> AM प्रणाली </td></tr><tr><td> पैराफिन तेल (भारी) </td><td> O122 - 1 </td><td> फिशर साइंटिफिक </td></tr><tr><td> के.एच. 2 पीओ 4 </td><td> P386-500 </td><td> फिशर साइंटिफिक </td></tr><tr><td> ना दो चार HPO </td><td> AC20651-5000 </td><td> फिशर साइंटिफिक </td></tr><tr><td> NaCl </td><td> BP3581 </td><td> फिशर साइंटिफिक </td></tr><tr><td> 4 MgSO </td><td> M80-500 </td><td> फिशर साइंटिफिक </td></tr></tbody></table>

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

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

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

मॉडल जीवों में, transgenesis जीन कार्यों में हेरफेर जबकि आरएनएआई विशिष्ट mRNA टेप 1-2 पछाड़ना सकते हैं कर सकते हैं. इस प्रोटोकॉल necromenic निमेटोड में stably प्रेषित डीएनए और क्षणिक डबल असहाय आरएनए परिचय की जरूरत तकनीक को वर्णन करना<em&gt; Pristionchus pacificus</em&gt; विकासवादी, विकास, और व्यवहार जीव विज्ञान में अध्ययन के लिए.

आरएनएआई जीन नॉकडाउन और Transgenesis मध्यस्थता Microinjection द्वारा Necromenic निमेटोड में 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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RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

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