RNAi Alimentación en cultivo líquido: Un método de alto rendimiento para derribar la expresión génica en C. elegans
Published: April 30, 2023
Abstract
Fuente: Groh, N. et al. Métodos para estudiar los cambios en la agregación de proteínas inherentes con edad en Caenorhabditis elegans. J. Vis. (2017).
RNAi es una poderosa herramienta genética disponible para los investigadores de C. elegans. Este video introduce un método para tratar más gusanos con RNAi que la alimentación de placas mediante la utilización de condiciones de cultivo líquido.
Protocol
Este protocolo es un extracto de Groh et al, Métodos para estudiar los cambios en la agregación de proteínas inherentes con edad en Caenorhabditis elegans, J. Vis. Exp. (2017). 1. Cultivo líquido de animales sin gónada para recoger animales jóvenes y envejecidos sometidos a RNAi dirigidos a un gen de interés …
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).