액체 배양에 RNAi 공급: C. elegans에서 유전자 발현을 녹여 고처리량 방법

Published: April 30, 2023

Abstract

출처: 그로, N.. Caenorhabditis elegans에서나이와 내재된 단백질 집계의 변화를 연구하는 방법J. 비스 익스펙. (2017).

RNAi는 C. elegans 연구원에 사용할 수 있는 강력한 유전 도구. 이 비디오는 액체 배양 조건을 활용하여 플레이트 공급보다 RNAi로 더 많은 벌레를 치료하는 방법을 소개합니다.

Protocol

이 프로토콜은 그로 외에서발췌, Caenorhabditis elegans에서나이와 고유 단백질 집계의 변화를 연구하는 방법, J. Vis. Exp. (2017). 1. 관심 유전자를 대상으로 RNAi를 대상으로 하는 젊고 숙성된 동물을 수집하는 고나드없는 동물의 액체 문화 <strong cla…

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]

Play Video

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).

View Video