RNAi-voeding in vloeibare cultuur: een methode met hoge doorvoer om genexpressie in C. elegans neer te slaan
Published: April 30, 2023
Abstract
Bron: Groh, N. et al. Methoden om veranderingen in inherente eiwitaggregatie met leeftijd in Caenorhabditis elegans te bestuderen. J.Vis. Exp. (2017).
RNAi is een krachtig genetisch hulpmiddel dat beschikbaar is voor onderzoekers van C. elegans. Deze video introduceert een methode om meer wormen met RNAi te behandelen dan plaatvoeding door gebruik te maken van vloeibare kweekomstandigheden.
Protocol
Dit protocol is een uittreksel uit Groh et al, Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans, J. Vis. Exp. (2017). 1. Vloeibare cultuur van geslachtsloze dieren om jonge en oudere dieren te verzamelen die onderworpen zijn aan RNAi gericht op een interessant gen <strong c…
Materials
| Fernbach culture flask | Corning | 4425-2XL | Pyrex, Capacity 2,800 ml, with 3 baffle indents | yes |
| Membrane Screw Cap | Schott | 1E+06 | GL45 | yes |
| Nutating Mixer | VWR | 444-0148 | yes | |
| Separatory funnel | Nalgene | 4300-1000 | Capacity 1,000 ml | no |
| 1 ml syringe | BD Plastipak | 3E+05 | no | |
| Gray needle, 27 G x ½ ", 0.4 mm x 13 mm | BD Microlance 3 | 3E+05 | no | |
| Membrane filters 0.025 µM | Millipore | VSWP04700 | no | |
| pH strip | Machery-Nagel | 92110 | pH-Fix 0-14 | no |
| Protease Inhibitor Cocktail | Roche | 5E+09 | Complete Mini EDTA-free tablets | no |
| Octoxynol-9 | Applichem | A1388 | Triton X-100 | no |
| 4-Morpholineethanesulfonic acid (MES) | Sigma-Aldrich | M1317 | no | |
| Nonylphenylpolyethylenglycol | Applichem | A1694 | Nonidet P40 (NP40) | no |
| DNaseI | Roche | 5E+09 | recombinant, RNase free | no |
| RNaseA | Promega | A7973 | solution | no |
| Total protein blot staining | Thermofisher | S11791 | Sypro Ruby protein blot stain | no |
| Total protein gel staining | Thermofisher | S12001 | Sypro Ruby protein gel stain | no |
| TCEP (tris (2-carboxyethyl) phosphine hydrochloride) | Serva | 36970 | no | |
| Iodoacetamide | Serva | 26710 | no | |
| Ammoniumbicarbonate | Sigma-Aldrich | 9830 | no | |
| Sequencing Grade Modified Trypsin | Promega | V5111 | no | |
| Isobaric tags for relative and absolute quantitation | Sciex | 4E+06 | iTRAQ Reagents Multiplex Kit | no |
| Centrifuge Avanti J-26XP | Beckmann Coulter | 4E+05 | yes | |
| Ultracentrifuge Optima Max-XP | Beckmann Coulter | 4E+05 | no | |
| Centrifuge 5424R | Eppendorf | 5E+09 | yes | |
| Centrifuge 5702 | Eppendorf | 6E+09 | yes | |
| Centrifuge Megafuge 40R | Thermo Scientific | 8E+07 | no | |
| Concentrator Plus | Eppendorf | 5E+09 | Centrifugal evaporator | no |
| Fluorescent stereo-microscope M165 FC | Leica | With Planapo 2.0x objective | no | |
| Dissection microscope | Leica | Leica S6E | yes | |
| High magnification microscope Zeiss Axio Observer Z1 | Zeiss | With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm | no | |
| Software | no | |||
| Image analysis software | ImageJ | no | ||
| Analysis of mass spectrometry data | Protein Prospector | http://prospector.ucsf.edu/prospector/mshome.htm | no | |
| E.coli strains | ||||
| OP50 | CGC | |||
| RNAi bacteria | ||||
| L4440 | Julie Ahringer RNAi library | |||
| C. elegans mutants | ||||
| CF2253 | CGC, strain name: EJ1158 | Genotype: gon-2(q388) | ||
| C. elegans transgenics | ||||
| DCD214 | Della David's lab at DZNE Tübingen | Genotype: N2; uqIs24[Pmyo-2::tagrfp::pab-1] | ||
| DCD215 | Della David's lab at DZNE Tübingen | Genotype: daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1] | ||
Citar este artigo
RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans. J. Vis. Exp. (Pending Publication), e20139, doi: (2023).