SO: Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft
KT: Methodology, Resources
NH: Conceptualization, Supervision, Writing - review & editing
AS: Conceptualization, Funding acquisition, Supervision, Writing - review & editing
The gonochoristic nematode
A. Plasmids for microparticle bombardment. They contain transgenes and the hygromycin resistance cassette.
Microparticle bombardment using DNA-coated gold particles (microcarrier) has been used to generate low-copy number transgene integrants in various nematodes, including
The plasmids for the bombardment were designed to contain a transgene and the hygromycin resistance gene cassette (Fig. 1A). Because of the high level of sequence conservation between
The protocol for microparticle bombardment and hygromycin B screening for
Using this method, we generated three
These results demonstrated that microparticle bombardment with hygromycin B screening is useful for transgenesis in the gonochoristic nematode
The wild-type of
Each plasmid for microparticle bombardment contains a transgene and the hygromycin B resistance gene cassette. The pTK89 plasmid for the expression of GFP::H2B histone was constructed by inserting the hygromycin B resistance gene cassette from pDD282 (a gift from Bob Goldstein, Addgene plasmid #66823) (Dickinson et al., 2015) and the transgene (
For the fluorescence imaging, ORCA-R2 Digital CCD camera (Hamamatsu Photonics) on Axioplan 2 imaging microscope (ZEISS) with Zeiss Plan-NEOFLUAR 10x/0.30 objective lens (ZEISS) was used. For the imaging of gonads and embryos, ORCA-R2 Digital CCD camera (Hamamatsu Photonics) on IX71 microscope (Olympus) with UPlanSApo 60x/1.30 silicone oil objective lens (Olympus) and a CSU-X1 spinning disc confocal system (Yokogawa Electric Corporation) was used.
This microparticle bombardment protocol corresponding to the 5 shots for a single plasmid, is based on the ones developed for other nematode species
1. Place the 10 mated
2. ~7 days later, wash out the worms from all plates with 5 ml of M9 buffer (22 mM KH 2 PO 4 , 42.3 mM Na 2 HPO 4 , 85.6 mM NaCl, 18.7 mM NH 4 Cl, 1 mM MgSO 4 ) per plate, and collect them into 50 ml conical tubes.
3. Stand the tubes for 10 minutes at room temperature and let most adult worms settle down.
4. Remove the supernatant containing bacteria, eggs, and larvae using a sterile Pasteur pipette.
5. Collect the precipitated adult worms into a single 50 ml conical tube by decantation.
6. Fill up the tube with M9 buffer.
7. Stand the tube for 10 minutes at room temperature and let most adult worms settle down.
8. Remove the supernatant.
9. Repeat washing steps (6 to 8) at least three times until the buffer appears clear of bacteria, eggs, and small larvae.
10. Transfer the adult worms to a 15 mL conical tube.
11. Centrifuge the tube for 1 minute at 800 × g to precipitate the adult worms and remove the supernatant. At this step, 2-5 ml worm suspension can be obtained. Use 1 ml of the worm suspension (2,000 ~ 20,000 worms) for one shot of bombardment.
12. Keep the worm suspension in the 15 mL conical tube at 22 °C horizontally on the shaker at 100 rpm for aeration until bombardment (The incubation period should not exceed 12 hours).
1. Weigh 35-50 mg of 1.6 μm Gold Microcarriers (Bio-Rad, 165-2264) into a 1.5 ml siliconized tube and vortex.
2. Add 1 ml of 70% ethanol and vortex for 5 minutes.
3. Stand for 15 minutes at room temperature.
4. Centrifuge the tube briefly and remove the supernatant.
5. Add 1ml of distilled water and vortex for 1 minute.
6. Stand for 1 minute at room temperature.
7. Centrifuge the tube briefly and remove the supernatant.
8. Repeat steps 4-6 twice to wash the gold microcarriers.
9. Suspend the gold microcarriers in 500 µl of 50% glycerol and store at 4 °C. (The gold microcarriers in 50% glycerol could be stored for 2 weeks.)
1. Vortex the gold microcarriers for 5 minutes.
2. Immediately, transfer 400 µl of the gold microcarriers in 50% glycerol to a new siliconized 1.5 ml tube and vortex for 1 min.
3. Add 40 µl of 2 μg/µl DNA, 400 µl of 2.5 M CaCl 2 , and 160 µl of 0.1 M spermidine.
4. Vortex for 2 minutes.
5. Stand for 1 minute at room temperature.
6. Centrifuge the tube briefly and remove the supernatant.
7. Add 1120 µl of 70% EtOH and gently tap the tube to resuspend the precipitate of the gold microcarriers.
8. Centrifuge the tube briefly and remove the supernatant.
9. Add 1120 µl of 100% EtOH and gently tap the tube to resuspend the precipitate.
10. Centrifuge the tube briefly and remove the supernatant.
11. Add 400 µl of 100% EtOH and gently tap the tube to resuspend the precipitate.
12. Use the gold microcarriers immediately for microparticle bombardment.
1. Microparticle bombardment was performed using Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, 165-2257) with Hepta Adaptor (Bio-Rad, 165-2225), 1550 psi Rupture Disks (Bio-Rad, 165-2331), and Hepta Stopping Screens (Bio-Rad, 165-2226).
2. Continue vortexing the gold microcarriers until the end of microparticle bombardment.
3. Wash 35 microcarrier disks (Macrocarriers (Bio-Rad, 165-2335), 7 disks for one shot) with 70% EtOH and 100% EtOH, and dry naturally.
4. Load 6 µl of the gold microcarriers on each of seven microcarrier discs and dry them until it discolors.
5. Soak 1550 psi Rupture Disks (Bio-Rad, 165-2331) in the isopropanol and dry naturally.
6. Spread 1 ml of the worm suspension uniformly onto a well-dried and -cooled NGM 90 mm plate.
7. Carry out the microparticle bombardment according to the manufacturer’s instructions with evacuating the bombardment chamber to 27 In. of Hg.
1. Add 10 ml of M9 buffer onto each bombarded plate and suspend the worms.
2. Dispense 10 ml of the worm suspension onto 10 dried HT115 seeded NGM 90 mm using a siliconized tip.
3. Incubate the plates at 27 °C for one day so that the bombarded worms will mate for propagation.
1. One day after microparticle bombardment, add 10 mg/ml hygromycin B (Invitrogen) onto the plates to a final concentration of 330 μg/ml and dry the plates.
2. Dissolve one unit of OP50 V.2 powder (LabTIE) in 25 ml S medium (100 mM NaCl, 5.74 mM K 2 HPO 4, 44.09 mM KH 2 PO 4 , 0.055 mM disodium EDTA, 0.025 mM FeSO 4 ・7 H 2 O, 0.010 mM MnCl 2 ・4 H 2 O, 0.010 mM ZnSO 4 ・7 H 2 O, 0.001 mM CuSO 4 ・5 H 2 O, 10 mM Potassium citrate pH 6.0, 3 mM CaCl 2 , 3 mM MgSO 4 , 5 mg/l cholesterol).
3. Add the 1 ml of OP50 suspension onto each plate and dry the plates.
4. Incubate the plates at 27 °C.
5. Four days after the bombardment, screen for survivors.
6. Add 5 ml of M9 buffer to each plate and let the surviving worms swim and float. Transfer the surviving worms to a new HT115-seeded NGM 90 mm plate using a sterile Pasteur pipette.
7. Incubate the plates at 27 °C for two days to let the worms grow into the adult stage.
8. Check the transgene expression in the adult worms with a fluorescent dissecting microscope.
9. Isolate the fluorescent-positive worms as integrant candidates.
10. Add wild-type adults (both females and males) onto each plate for mating (female: at least 5 worms, male: five times as many as females)
11. Incubate at 27 °C for propagation.
12. After propagation, repeat hygromycin B selection for confirmation.
6. Keep incubating the plates at 27 °C.
7. ~three weeks later, isolate the surviving worms as integrant candidates and check the transgene expression with a fluorescent microscope.
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SA1161 |
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This work |
SA1393 |
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This work |
SA250 |
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CGC (Toya
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NKZ35 |
Wild-type |
CGC |
SA1438 |
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This work |
SA1555 |
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This work |
SA1648 |
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This work |
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pTK89 |
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Promoters, ORF, and 3’UTR derived from
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pTK68 |
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Promoters and 3’UTR derived from
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pSO5 |
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Promoters, ORF, and 3’UTR derived from
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This work was supported by JST, CREST Grant Number JPMJCR18S7, Japan to AS, and JST SPRING, Grant Number JPMJSP2114 and MEXT's University Fellowship Founding Project for Innovation Creation in Science and Technology to SO.