RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli

Abstract

Source: Kolundzic, E., 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).

This video describes the principles behind RNAi treatment in C. elegans and demonstrates a protocol to knockdown lin-35 in a transgenic worm strain.

Protocol

This protocol is excerpted from 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).

1. Solution Preparation
   1. NGM-Agar plates (1 L)
      1. Add 3 g of NaCl, 2.5 g of peptone media (e.g., Bacto-Peptone), and 20 g of agar. After autoclaving, add 1 mL of cholesterol (5 mg/mL in 95% EtOH stock), 1 mL of 1 M MgSO₄, 1 mL of 1 M CaCl2, 25 mL of 1 M K2PO

Results

Figure 1: Pvul phenotype caused by RNAi against lin-53. (Top) DIC picture of L4/young adult stage F1 progeny worms derived from control or lin-53 RNAi treated mothers. Scale bars = 20 µm. (Bottom) Animals treated with lin-53 RNAi display the p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
<strong>Chemicals&nbsp;</strong>
Agar-Agar, Kobe I	Roth	5210.1
Bactopeptone&nbsp;	A. Hartenstein GmbH	211 677	Peptone Media&nbsp;
Yeast extract&nbsp;	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&nbsp;	3904.2
MgSO4	VWR	25,163,364
Na2HPO4	Roth	P030.1
NaCl	Roth	9265.1
NaClO	Roth	9062.3
NaOH (5 N)	Roth&nbsp;	KK71.1
Tris	Roth	AE15.2
Carbenicillin	Roth	634412
Tetracycline	Roth	2371.2
<strong>Incubators</strong>
Incubator&nbsp;	Sanyo MIR-5	5534210	for maintenance of worm strains at 15 &deg;C or 25 &deg;C
<strong>Microscopes</strong>
Stereomicroscope	SMZ745	Nikon
<strong>Bacterial strains</strong>
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&nbsp;			Uracil auxotroph E. coli strain
<strong>Worm strains</strong>
BAT28:&nbsp;otIs305 (hsp16.2prom::che-1::3xHA) ntIs1 (gcy-5prom::gfp) V.			derived from OH9846 by 4x times more back crossing with N2
<strong>RNAi Clones</strong>
lin-53	SourceBioscience	ID I-4D14	Ahringer library 16B07 ChromI, K07A1.12
empty vetor: L4440	Addgene&nbsp;	#1654