Cell Fate Reprogramming of Nematode Germ Cells into Neurons

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 demonstrates cell fate reprogramming of nematode germ cells into neurons. Transgenic larvae of the nematode Caenorhabditis elegans are subjected to RNA interference (RNAi) to knock down a specific chromatin-regulating factor, making the germ cells of their progeny more susceptible to cell fate reprogramming. Heat shock is applied to the progeny, inducing the germ cells to express a neuron-specific transcription factor that triggers their transformation into neurons, which is identified using an expressed fluorescent reporter.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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% ethanol [EtOH] stock), 1 mL of 1 M MgSO₄, 1 mL of 1 M CaCl₂, 25 mL of 1 M K₂PO₄, and 1 mL of amphotericin B (2.5 mg/mL stock).
2. NGM-Agar RNAi plates (1 L)
   1. Add 3 g ....

Results

Figure 1: pvul phenotype caused by RNAi against lin-53. (Top) Differential interference contrast (DIC) picture of L4/young adult stage F1 progeny worms derived from control or lin-53 RNAi treated mothers. Scale bars = 20 µm. (Bottom) Ani.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Agar-Agar, Kobe I	Roth	5210.1
Bactopeptone	A. Hartenstein GmbH	211 677	Peptone Media
Yeast extract	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	3904.2
MgSO4	VWR	2,51,63,364
Na2HPO4	Roth	P030.1
NaCl	Roth	9265.1
NaClO	Roth	9062.3
NaOH (5 N)	Roth	KK71.1
Tris	Roth	AE15.2
Carbenicillin	Roth	634412
Tetracycline	Roth	2371.2
Incubators
Incubator	New Brunswick Scientific C24	M1247-0052	for heat-shock
Incubator	Sanyo MIR-5	5534210	for maintenance of worm strains at 15 °C or 25 °C
Microscopes
Fluorescence microscope	M205 FA	Leica
Microscope (Imaging) + camera	Axio Imager 2	Zeiss
Microscope (Imaging) + camera	Sensicam	PCO
Stereomicroscope	SMZ745	Nikon
Bacterial strains
Escherichia coli HT115			F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse III minus).
Escherichia coli OP50			Uracil auxotroph E. coli strain
Worm strains
BAT28: otIs305 (hsp16.2prom::che-1::3xHA) ntIs1 (gcy-5prom::gfp) V.			derived from OH9846 by 4x times more back crossing with N2
RNAi Clones
lin-53	SourceBioscience	ID I-4D14	Ahringer library 16B07 ChromI, K07A1.12
empty vetor: L4440	Addgene	#1654
Misc.
Worm pick			self-made using a pasteur pipette and a platinum wire