ND-labeling C. elegans worms

ND-labeling C. elegans worms protocol in 10.1126/sciadv.aba9636

Simo Sun, Eriko Kage-Nakadai, Masazumi Fujiwara

Mar. 30, 2021

We used a traditional and well-established method which has been used to generate transgenic C. elegansin molecular biology experiments (1). There is an excellent protocol uploaded to bio-protocol by Rieckher and Tavernarakis (2) ( https://bio-protocol.org/e2565 ). Our protocol starts from Step 6 of Procedure A with nanodiamond (ND) instead of DNA and ends at Procedure C in Rieckher’s protocol. We describeour protocol focusing on our modification made, as below.

Material

1. The wild-type C. elegans strain Bristol N2 was obtained from the Caenorhabditis Genetics Center (Minneapolis, MN)
2. Polyglycerol-grafted nanodiamond (PG-ND) suspension (1.88 g/mL) was prepared in the manner described by Zhao et al. (3)

Equipment and Regent

1. 2% agarose pads
2. Vortex mixer
3. M9 buffer
4. Wormpicker with platinum wire and pre-flattened tip
5. Microinjection glass capillaries (Narishige, ND-1)
6. Pipette puller (Narishige, PC-100)
7. Paraffin oil (Wako, Japan)
8. Micromanipulator (Narishige, MN-4)
9. Microinjection system (Narishige, IM-31)
10. Microscope used for the injection was equipped with differential interference contrast (Olympus,IX73)
11. E. coli-seeded NGM culture

Procedure

   1. Prepare glass needle using microinjection glass capillary and pipette puller following the manufacturer’s manual

   2. Resuspend PG-ND solution for 15 min, using vortex mixer (or sonication if necessary). Dilute them to less than 1.88 g/mL with M9 buffer (if necessary).

   3. Load < 0.5 μL of PG-ND dispersion into prepared glass needle (step 1). Set it into the needle holder of the micromanipulator.

   4. Because the diameter of tip of glass needle is too narrow for PG-ND to go through, we had to open the tip of glass needle manually. We just asked the tip of needle touch the board of slide glass extremely gently and quickly (Figure 1), under the control of micromanipulator. This step probably needs some skills, a little patience and luck.

   5. Place a drop of about 50 µL paraffin oil on a 2% agarose pad. Under the stereoscope, transfer 1 worm into theparaffin oil.

   6. Place the agarose pad on the gliding table of the microscope for microinjection and swiftly commencewith the Injection procedure (https://bio-protocol.org/e2565).

   7. Incubate the worms on E. coli-seeded NGM culture overnight in 20^oC to ask them recover from damages.

References

1. C. C. Mello, J. M. Kramer, D. Stinchcomb, V. Ambros, Efficient gene transfer in C. elegans: Extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959– 3970 (1991).
2. Rieckher, M. and Tavernarakis, N. (2017). Generation of Caenorhabditis elegans TransgenicAnimals by DNA Microinjection. Bio-protocol 7(19 ): e2565. DOI: 10.21769/BioProtoc.2565.
3. L. Zhao, T. Takimoto, M. Ito, N. Kitagawa, T. Kimura, N. Komatsu, Chromatographic separation of highly soluble diamond nanoparticles prepared by polyglycerol grafting. Angew. Chem. Int. Ed. 50, 1388–1392 (2011).