Generering av Transgena C. elegans Genom Biolistic Transformation
Summary
Transgena maskar används ofta i<em> C. elegans</em> Forskning. Som beskrivs är en enkel men effektiv, protokoll att införa transgener till maskarna med hjälp biolistic bombardemang med DNA-belagda guldpartiklar. Arbetet med och resultaten av bombardemang jämförelse med mikroinjektion för generering av transgena djur.
Abstract
The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.
Protocol
Discussion
Biolistic bombardemang är en enkel metod att introducera främmande DNA i många organismer, inklusive C. elegans 1,5,6,7,16. Det är beroende av guldpartiklar bildar ett komplex med DNA i närvaro av CaCl 2. Katjoniska polyaminer, såsom spermidine, skydda DNA från nukleas nedbrytning in vivo. Eftersom spermidine är ett instabilt molekyl, är det viktigt att förvara det i små alikvoter vid -20 ° C, och göra lösningen rätt innan du utför bombardemanget. Som vi beskrev i beskjutni…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Detta arbete stöddes av såddkapitalfonder från University of Pittsburgh och NIH bevilja AG028977 till ALF
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Gold particles | Inbio gold | BD021 | 1micron | |
| Midiprep kit | Qiagen | 12143 | ||
| Spermidine | Sigma | S4139 | ||
| Protamine | Sigma | P4505 | ||
| Macrocarriers | Biorad | 165-2335 | ||
| PDS-1000/He Hepta System | Biorad | 165-2257 | ||
| Rupture disk | Biorad | 165-2330 | ||
| Nystatin | Sigma | N1638 | ||
| Streptomycin | MP | 100556 |
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