RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans

Published: April 30, 2023

Abstract

Quelle: Groh, N. et al. Methoden zur Untersuchung von Veränderungen in der inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegansJ. Vis. (2017).

RNAi ist ein leistungsstarkes genetisches Werkzeug, das C. elegans Forschern zur Verfügung steht. Dieses Video stellt eine Methode vor, um mehr Würmer mit RNAi zu behandeln als Plattenzuführen, indem flüssige Kulturbedingungen verwendet werden.

Protocol

Dieses Protokoll ist ein Auszug aus Groh et al, Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans, J. Vis. Exp. (2017). 1. Flüssige Kultur von gonadenlosen Tieren zur Sammlung von jungen und gealterten Tieren, die RNAi ausgesetzt sind und auf ein Gen von Interesse abzielen …

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]

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Cite This Article
RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans. J. Vis. Exp. (Pending Publication), e20139, doi: (2023).

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