Embryo microinjection of the lecithotrophic sea urchin Heliocidaris erythrogramma
2School of Medical Science and Bosch Institute, Department of Anatomy and Histology, The University of Sydney, Sydney, NSW 2006, Australia
3School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia
4Center for Genomic and Computational Biology, Duke University, Durham, NC 27708, USA
- Filtered natural sea water (FSW)
- Agarose (Sigma, cat. #: A9539)
- Gelatin (Sigma, cat. #: G1890)
- Potassium chloride (Sigma, cat. #: P9541)
- Ficoll 400 (Sigma cat. #: F-9378)
- Molecular grade glycerol (Sigma cat. #: G5516)
- Fixable tetramethylrhodamine-dextran (TMR) 10000 MW (Sigma cat. #: 73766)
- Penicillin-streptomycin 100× stock solution: 10000 units penicillin and 10 mg streptomycin/ml (Sigma cat. #: P4333)
- RNase-free water (not DEPC-treated)
- Experimental reagent(s) of choice, e.g., IVT-mRNA, morpholino oligos, CRISPR guides and Cas9
- 0.1% gelatin, 0.05% paraformaldehyde (to coat tools)
- 2× microinjection solution (RNase-free/molecular-grade reagents), for 20 μl:
- 3.5 μl water
- 6.5 μl lysine-fixable fixable tetramethylrhodamine (TMR) dextran stock, 10000 MW (150 mg/ml) (ThermoFisher D1868)
- 2.0 μl 4M KCl (Sigma cat. #: 60128)
- 8.0 μl glycerol (Sigma cat. #: G5516)
- Pasteurized filtered sea water (PFSW): Coarsely filter natural sea water through a gravity-fed glass filter. Pasteurize at 65°C 30 min at temperature (heated stir plate or water bath) and returned to room temperature. Alternatively, vacuum filter with 0.22 μm filter.
- Spawning injection solution: 0.5 M KCl (Sigma cat. #: P9333)
- 1× Penicillin-Streptomycin in PFSW
- 2% w/v Ficoll 400 in PFSW
- Embryo-dedicated labware:
- 1 L glass bottles (VWR cat. #: 10754-820)
- 500 ml beakers (VWR cat. #: 10754-956)
- 250 ml flask (VWR cat. #: 10536-914)
- Stir bars (VWR cat. #: 58948-138)
- Lidded plastic boxes
- 10 ml syringe with needle
- Heated stir plate
- Microwave oven
- Needle puller (Sutter P97 Flaming/Brown Micropipette Puller) equipped with standard 2.5 × 2.5 mm box filament
- Microinjector (General Valve Picospritzer II)
- Nitrogen tank and regulator
- Miniature ruler (Ted Pella 13623) or micrometer
- Dissecting microscope (Olympus SZH) with white light
- Epifluorescence dissecting microscope with correct light source and fluorescence filter set for the selected dye
- P10 pipettor and tips (RNase-free if injecting mRNA)
- Humid chambers (dedicated airtight lidded plastic boxes with moistened absorbent material such as a sponge)
- Coarse filters for FSW (Millipore AP25 prefilters AP2504700)
- 60 × 15 mm petri dish lids (Falcon 351007)
- 4 well IVF dishes (Multidish Polystyrene 4 Well Nunclon Round w/Lid Sterile 66 × 66 MD4 for IVF 15 mm Well) (Thermo 176740)
- 6 well plates (e.g., Falcon 351146), coated with gelatin or Corning® Costar® Ultra-Low Attachment 6-well plates
- Plastic transfer pipettes
- 5” glass Pasteur pipettes
- Pasteur pipette bulbs
- Latex tubing
- Needle stock: thin wall borosilicate glass capillaries with inner filament, OD 1.0 mm, ID 0.75 mm (World Precision Instruments TW100F-6)
- 150 micron nitex filter basket
- Inject under a dissecting microscope with a black background.
Prepare tools and reagents
1.Pull needles using the following parameters: heat = 760 (100% ramp value), pull = 80, delay = 120. The needle’s taper should begin approximately 9 mm from the tip. The tip of a representative needle is shown in Figure 1.
2.Gelatin-coat 6-well plates to use as culture dishes (or use IVF dishes). Fire-polish and gelatin-coat glass Pasteur pipettes to transfer embryos.
3.Filter, pasteurize, and cool to room temperature natural sea water.
4.Prepare injection dishes.
4.1.Make 2% agarose in 50% FSW and melt gently. Into the lids of 60 mm petri dishes, pour just enough agarose to cover the surface of each overturned lid. Allow agarose to solidify.
4.2.Fill injection dishes with PFSW for at least 1 h before use. Store dishes filled with PFSW and protected from dust, at room temperature. Use within 24 h.
5.Spawn female and male urchins by injecting ~0.5–1 ml 0.5 M KCl into an adult urchin using a syringe fitted with a 24-gauge hypodermic needle.
6.Collect sperm dry into a microfuge tube and store at 4°C for up to 1 week. Sperm are activated in the presence of seawater and the sperm will be compromised if any gets into the tube.
7.Collect eggs. Allow female to spawn at the bottom of a beaker of coarsely filtered seawater (Fig. 1). With a transfer pipette, move the eggs from the top of the beaker to a clean 500 ml beaker of FSW.
8.Wash and partially de-jelly eggs by washing 3× with ~500 ml FSW. Draw off FSW with a bulb syringe through a basket made with 150 µm mesh attached to either a plastic beaker with the bottom removed or a small section of PVC pipe. Allow eggs to rise to surface after last wash.
9.Transfer washed eggs from surface into a small volume (~50–100 ml) PFSW in a 500 ml container. Dilute sperm by adding 500 µl FSW to 5 µl sperm. Fertilize washed eggs with diluted sperm.
10.After 5–10 min, check under magnification for fertilization envelopes to confirm fertilization. The perivitelline space tends to be relatively small in this species, so look for a shiny surface. If most do not have visible fertilization envelopes at 5 min, add more sperm to synchronize the time of fertilization.
11.After confirming fertilization, wash out excess sperm by filling dish with PFSW. Allow zygotes to rise to the top, and transfer into a clean dish of PFSW.
Set up injection dishes
12.Pick best-looking fertilized zygotes into a clean dish filled with pasteurized FSW for storage until injected.
13.Transfer 20–50 embryos into an injection dish containing 2% w/v Ficoll 400 in PFSW. Keep approximately constant the number of embryos per plate for the experiment and simply inject more plates to produce more experimental embryos.
14.Draw off the solution and replace with fresh 2% Ficoll-PFSW. Swirl the dish to bring zygotes to float near the dish’s center (Fig. 3A).
15.Draw off Ficoll-PFSW until zygotes make firm contact with agarose pad (Fig. 3B and 3C). Wait 5–10 s, and then gently add Ficoll-PFSW dropwise to each cluster of zygotes until the meniscus is beyond their cell membranes. If they float away, the volume is too large; draw it off and try again.
16.Load needles with 1–2 µl reagent of choice in 1× injection mix. Use pipettor to place solution on the back end of the needle, where the microcapillary will draw it to the sharpened end.
17.Turn on microinjector and gas supply. Set the Picospritzer air pressure to 30 PSI. Use the constant pressure setting until the needle is broken.
18.Load a needle into the microinjection rig. Under dissecting microscope, break needle on the ridges on the edge of the injection plate. Alternatively, forceps or a hypodermic needle may be used to break open the needle.
19.Switch to the pulse setting on the Picospritzer. Check that the microinjector settings and needle opening create the desired bolus size at ~100 ms pulse duration. The ideal bolus is no greater than 1/10 the embryo volume. Adjust the pressure duration setting accordingly.
20.Approach at a 30°–45° angle so that the needle enters near a relatively large volume of Ficoll-PFSW. Insert the needle 1/5–1/3 diameter of the egg. The dye will rapidly spread through the embryo if it is properly injected (Fig. 4; Video S1).
21.Immediately after injecting the whole plate, flood with Ficoll-PFSW and place in a humid chamber.
Pick and culture injected embryos; complete assays
22.Sort experimental embryos between 4-cell stage and early blastula (Fig. 4D). Transfer injected embryos from injection dish to culture dish with gelatin-coated pipette. Under a fluorescent dissecting microscope, transfer evenly injected, similarly bright embryos (Fig. 5) and transfer into coated culture dishes (IVF plates or gelatin-coated multi-well plates) containing 1× Penicillin-Streptomycin in PFSW (Pen-Step PFSW).
23.Culture embryos in a humid chamber at 17°C–22°C in Pen-Step PFSW. Change water and remove dead embryos twice daily. Culture density for first 24 h should not exceed 10 embryos/ml.
24.Fix appropriately for the intended assay at a predetermined time point.
|10||eggs fail to fertilize||sperm concentration too low||add more sperm|
|10||eggs fail to fertilize||sperm not viable||collect fresh sperm; always store sperm dry|
|10||eggs fail to fertilize||eggs not viable||spawn another female|
|15||uninjected zygotes spontaneously rupture||too much water removed||set up a new injection dish removing less water; work quickly after removing water|
|20||injected zygotes rupture||needle too large||break fresh needle closer to tip|
|20||injected zygotes leak||Ficoll % too low||follow steps 13–14 exactly|
|20||injected zygotes leak||needle too large||break fresh needle closer to tip|
|20||visible spots of dye remain at zygote surface||needle too large||break fresh needle closer to tip|
|20||visible spots of dye remain at zygote surface||needle not inserted deep enough||insert the needle 1/5–1/3 diameter of the egg|
|20||needle clogs||occasional needle clogs are unavoidable||use "clear" function on microinjector; gently wipe needle on agarose pad; re-break needle|
|20||needle clogs||needle bore too large||break fresh needle closer to tip|
|20||needle clogs||needle not removed from zygote immediately after injection||pull out needle immediately after injecting the bolus|
|22||abnormal cleavage planes||polyspermy||fertilize again using less sperm in step 9 and/or washing more in step 11|
|23||injected embryos die at much higher rate than uninjected control||too much material injected||reduce bolus size|
|23||injected embryos die at much higher rate than uninjected control||infection||change water more often|
|23||experimental embryos die at much higher rate than injection control||reagent is lethal at current dose||titrate reagent dose|
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