आरएनएआई जीन नॉकडाउन और Transgenesis मध्यस्थता Microinjection द्वारा Necromenic निमेटोड में Pristionchus pacificus</em
Summary
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> Pristionchus pacificus</em> विकासवादी, विकास, और व्यवहार जीव विज्ञान में अध्ययन के लिए.
Abstract
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.
Protocol
Discussion
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Product: | Catalog #: | Supplier: |
|---|---|---|
| DMI3000 Injection microscope | DMI3000 | Leica |
| Microinjector manipulator (Direct drive) | Narashige BC-3 Ball joint | Tritech Research |
| Needle Puller | Narashige PC-10 | Tritech Research |
| MicroInjector™ All-Digital Multi-pressure System | Narashige MINJ-D | Tritech Research |
| GeneElute™Mammalian Genomic DNA Miniprep Kit | G1N70 | Sigma |
| pJet Cloning Jet kit | K1231 | Fermentas |
| GeneJET plasmid Miniprep kit | K0502 | Fermentas |
| DNA Clean & Concentrator™ | D4005 | ZYMO Research |
| BLOCK-IT™ RNAi TOPO® transcription kit | K3500-01 & K3650-01 | Invitrogen |
| Difco™Agar Noble | DF0142-15-2 | Fisher Scientifi |
| Microscope cover glass (1.5 – 0.16 to 0.19mm thick; Size: 50 x 45mm) | 12-554-F | Fisher Scientific |
| Glass capillaries (filament) | 615000 | A-M systems |
| Paraffin Oil (Heavy) | O122-1 | Fisher Scientific |
| KH2PO4 | P386-500 | Fisher Scientific |
| Na2HPO4 | AC20651-5000 | Fisher Scientific |
| NaCl | BP3581 | Fisher Scientific |
| MgSO4 | M80-500 | Fisher Scientific |
References
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