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

Protocollo

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 K2PO₄, and 1 mL of amphotericin B (2.5 mg/mL stock).
   2. NGM-Agar RNAi plates (1 L)
      1. Add 3 g of NaCl, 2.5 g of peptone media, 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 K₂PO₄, 1 mL of amphotericin B (2.5 mg/mL stock), 1 mL of 1 M IPTG, and 1 mL carbenicillin (50 mg/mL).
   3. LB-Agar (1 L)
      1. Add 10 g of peptone media, 5 g of yeast extract, 5 g of NaCl, 20 g of agar, 10 mL of 1 M Tris pH 8.0. Add H₂O to 1 L. After autoclaving, add 1 mL of 50 mg/mL carbenicillin and 2.5 mL of 5 mg/mL tetracycline.
   4. LB Medium (1 L)
      1. Add 10 g of peptone media, 5 g of yeast extract, 5 g of NaCl, and 10 mL of 1 M Tris pH 8.0. Add H₂O to 1 L. After autoclaving, add 1 mL of 50 mg/mL carbenicillin.
   5. M9-buffer (1 L)
      1. Add 6.0 g of Na₂HPO₄, 3 g of KH₂PO₄, 5 g of NaCl, and 50 mg of gelatin. Before usage, add 1 mL of 1 M MgSO₄.
   6. Bleaching solution (10 mL)
      1. Add 1 mL of NaClO and 2 mL of 5 N NaOH. Add H2O to 10 mL.
2. Preparation of RNAi Plates
   NOTE: C. elegans is typically cultured in the laboratory on 6 cm Petri plates containing 7.5 mL of Nematode Growth Medium Agar (NGM-Agar). This protocol is optimized for worms kept at 15 °C. To prepare plates with NGM, use standard sterile techniques to prevent fungal and bacterial contamination. The following is the protocol to prepare NGM plates supplemented with reagents for RNAi.
   1. Prepare 6-cm NGM-Agar RNAi plates containing 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and 50 µg/mL carbenicillin. Let them dry for 24 – 48 h at room temperature in the dark¹².
      1. Keep RNAi plates at 4 °C in the dark. Do not use if older than 14 days.
   2. Select the RNAi bacteria (Escherichia coli HT115) clone containing the L4440 plasmid with the lin-53 gene DNA sequence from the frozen glycerol stock from the available RNAi library on LB-agar plates containing 50 µg/mL carbenicillin and 12.5 µg/mL tetracycline by using the three-phase streaking pattern. Grow the bacteria at 37 °C overnight. This clone allows IPTG-dependent production of lin-53 dsRNA in the bacteria.
      1. Additionally, grow RNAi bacteria that contain the L4440 plasmid without any gene sequence as the empty vector control.
   3. The next day, pick a single colony from lin-53 or empty vector LB-agar plates using a 200 µL pipette tip and inoculate each of them into a separate culture tube containing 2 mL of liquid LB medium supplemented with 50 µg/mL carbenicillin. Grow cultures overnight at 37 °C until they reach an optical density (OD) at 600 nm of 0.6 – 0.8. Measure the OD using a spectrophotometer.
      CAUTION: The liquid LB media should not contain tetracycline in contrast to the LB-agar plate used for streaking the bacteria from the glycerol stock, since inclusion of tetracycline during feeding leads to a decreased RNAi efficiency.
   4. Add 500 µL of each bacterial culture (lin-53 or empty vector) to 6 cm NGM-Agar RNAi plates using a multipipette. Incubate plates with a closed lid overnight at room temperature in the dark to dry. During this time, the IPTG in the NGM plates will induce production of dsRNA in the bacteria.
      1. Use at least 3 plates per bacterial culture per experiment. This will provide 3 technical replicates for each experiment.
   • Store dried NGM-agar RNAi plates with bacteria at 4 °C in the dark for up to two weeks.
3. Preparation of C. elegans Strain BAT28
4. Maintain the worm strain BAT28 containing the transgenes otIs305 [hsp-16.2prom::che-1, rol-6(su1006)] and ntIs1 [gcy-5prom::gfp] on OP50 bacteria using standard NGM-Agar plates at 15 °C.
   NOTE: For detailed protocol on Nematode culture, see. The rol-6(su1006) is a dominant allele and commonly used phenotypic injection marker¹⁶ that causes the typical rolling movement. It is used in the otIs305 transgene to track hsp-16.2prom::che-1.
   1. Keep the BAT28 strain at 15 °C at all times in order to prevent precautious activation of the hsp-16.2prom::che-1 transgene. Reduce exposure to temperatures higher than 15 °C while handling the strain as much as possible.
5. To age-synchronize worms, use the bleaching technique.
   1. Wash off 6 cm NGM-Agar plates containing adults and eggs of BAT28 using 900 µL M9 buffer. Pellet worms by centrifuging at 900 x g for 1 min. Remove the supernatant.
   2. Add 0.5 – 1 mL of bleaching solution and shake the tube for approximately 1 min until adult worms start to burst open. Monitor using a standard stereomicroscope.
   3. Pellet worms by centrifuging at 900 x g for 1 min. Remove the supernatant.
   4. Wash the worm pellet 3 times by adding 800 µL of M9 buffer and centrifuging at 900 x g.
   5. Place the cleaned eggs on fresh NGM-plates seeded with OP50 bacteria. Grow a bleached population on OP50 bacteria at 15 °C until they reach L4 stage (approximately 4 days). L4 larvae can be recognized by a white patch approximately halfway along the ventral side of the worm using a standard stereomicroscope.
      NOTE: To achieve the germ cell to neuron conversion phenotype upon depletion of lin-53, it needs to be depleted in the parental generation (P0). The scoring for the conversion phenotype is undertaken in the following generation (filial generation F1). To achieve the deplete lin-53 already in the P0, L4 animals are subjected to RNAi.
6. Manually transfer 50 L4 stage worms per replicate using a platinum wire to an NGM plate that does not contain any bacteria and let worms move away from any transferred OP50 bacteria. Let worms move on the plate for around 5 minutes. Use bacteria from the NGM-Agar RNAi plates previously seeded with lin-53 RNAi bacteria (or empty vector control) to transfer to the respective RNAi plates.
   1. Avoid transferring OP50 bacteria to the actual RNAi plates. Work fast to minimize exposure time of the strain to temperatures higher than 15 °C.
7. Incubate worms on NGM-Agar RNAi plates at 15 °C for approximately 7 days until F1 progeny of the worms reaches L3 – L4 stage. They can be clearly separated from the P0 animals since they are bigger and thicker than the F1 progeny.
   1. Visually check under a standard stereomicroscope whether F1 progeny worms show the protruding vulva (pvul) phenotype as shown in Figure 1. RNAi against lin-53 has pleiotropic effects and causes the pvul phenotype which can be used to assess whether RNAi against lin-53 has been successful.
      NOTE:  RNAi against lin-53 can also cause lethality of F1 embryos. This effect increases if plates are exposed to higher degrees than 15 °C before animals reached the L3 – L4 stage. Dead embryos can be recognized by arrested development and lack of hatching.
   2. Incubate plates in the dark since IPTG is light sensitive.

Risultati

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 protruding vulva (pvul) phenotype (black arrow-heads). The pvul phenotype confir...

Materiali

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	25,163,364
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	Sanyo MIR-5	5534210	for maintenance of worm strains at 15 °C or 25 °C
Microscopes
Stereomicroscope	SMZ745	Nikon
Bacterial strains
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			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