RNAi Plating for C. elegans Feeding: En teknik til at fremkalde Target dsRNA-udtryk i E. coli
Published: April 30, 2023
Abstract
Kilde: Kolundzic, E., et al. Anvendelse af RNAi og Heat-shock-induceret Transskription Factor Expression at omprogrammere kimceller til neuroner i C. elegans. J. Vis. Exp. (2018).
Denne video beskriver principperne bag RNAi behandling i C. elegans og viser en protokol til knockdown lin-35 i en transgen orm stamme.
Protocol
Denne protokol er uddrag fra Kolundzic, et al, Anvendelse af RNAi og Heat-shock-induceret Transskribering Factor Expression at omprogrammere kimceller til neuroner i C. elegans, J. Vis. Exp. (2018). Løsningsforberedelse NGM-Agar plader (1 L) Der tilsættes 3 g NaCl, 2,5 g peptonmedier (f.eks. Bacto-Peptone) og 2…
Representative Results
Figur 1: Pvul phenotype forårsaget af RNAi mod lin-53. (Øverst) DIC billede af L4/unge voksne fase F1 afkom orme stammer fra kontrol eller lin-53 RNAi behandlede mødre. Skalastænger = 20 μm. (Nederst) Dyr behandlet med lin-53 RNAi udviser den fremspringende vulva (pvul) fænotype (sorte pilehoveder). Den pvul phenotype bekræfter, at RNAi mod lin-53 var en…
Materials
| Chemicals | |||
| Agar-Agar, Kobe I | Roth | 5210.1 | |
| Bactopeptone | A. Hartenstein GmbH | 211 677 | Peptone Media |
| Yeast extract | AppliChem | A1552,0100 | |
| Amphotericin B | USBiological | A2220 | |
| CaCl2 | Merck Biosciences | 208290 | |
| Cholesterol | Roth | 8866.2 | |
| Gelatine | Roth | 4275.3 | |
| IPTG | Sigma | 15502-10G | |
| K2PO4 | Roth | T875.2 | |
| KH2PO4 | Roth | 3904.2 | |
| MgSO4 | VWR | 25,163,364 | |
| Na2HPO4 | Roth | P030.1 | |
| NaCl | Roth | 9265.1 | |
| NaClO | Roth | 9062.3 | |
| NaOH (5 N) | Roth | KK71.1 | |
| Tris | Roth | AE15.2 | |
| Carbenicillin | Roth | 634412 | |
| Tetracycline | Roth | 2371.2 | |
| Incubators | |||
| Incubator | Sanyo MIR-5 | 5534210 | for maintenance of worm strains at 15 °C or 25 °C |
| Microscopes | |||
| Stereomicroscope | SMZ745 | Nikon | |
| Bacterial strains | |||
| Escherichia coli HT115 | F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAseIII minus). | ||
| Escherichia coli OP50 | Uracil auxotroph E. coli strain | ||
| Worm strains | |||
| BAT28: otIs305 (hsp16.2prom::che-1::3xHA) ntIs1 (gcy-5prom::gfp) V. | derived from OH9846 by 4x times more back crossing with N2 | ||
| RNAi Clones | |||
| lin-53 | SourceBioscience | ID I-4D14 | Ahringer library 16B07 ChromI, K07A1.12 |
| empty vetor: L4440 | Addgene | #1654 |
Cite This Article
RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli. J. Vis. Exp. (Pending Publication), e20138, doi: (2023).