RNAi Plating voor C. elegans Feeding: Een techniek om doel dsRNA expressie te induceren in E. coli
Published: April 30, 2023
Abstract
Bron: Kolundzic, E., et al. Toepassing van RNAi en heat-shock-geïnduceerde transcriptiefactorexpressie om kiemcellen te herprogrammeren op neuronen in C. elegans. J.Vis. Exp. (2018).
Deze video beschrijft de principes achter de RNAi-behandeling bij C. elegans en demonstreert een protocol om lin-35 in een transgene wormstam neer te slaan.
Protocol
Dit protocol is overgenomen uit Kolundzic, et al, Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans, J. Vis. Exp. (2018). Oplossingsvoorbereiding NGM-Agar platen (1 L) Voeg 3 g NaCl, 2,5 g pepton media (bijv. Bacto-Peptone) en 20 g agar toe…
Representative Results
Figuur 1: Pvul fenotype veroorzaakt door RNAi tegen lin-53. (Boven) DIC-beeld van L4/young adult stadium F1-nakomelingenwormen afgeleid van controle of lin-53 RNAi behandelde moeders. Schaalbalken = 20 μm. (Onder) Dieren behandeld met lin-53 RNAi geven het uitstekende vulva (pvul) fenotype (zwarte pijlpunten) weer. Het pvul fenotype bevestigt dat RNAi tegen lin-53<…
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 |
Citar este artigo
RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli. J. Vis. Exp. (Pending Publication), e20138, doi: (2023).