1. Do not use DNA in 1X TE or other Tris-based buffers.

2. Several workers prefer to dechorionate the embryos before injection. This is done en masse with pronase (0.5 mg/mL) followed by extensive washes to remove the residual pronase before proceeding. However, it is not necessary, since the embryos can be injected through the chorion (1).

3. The embryos are best handled with a glass Pasteur pipet that has been cut and polished in a Bunsen flame to give an aperture of about 2.0 mm.

4. In The Zebrafish Book, Westerfield (1), describes a simple gas pressure injection apparatus that may be made by a competent technical workshop. Alternatively, a pneumatic picopump air pressure supply system is supplied World Precision Instruments.

5. The needle puller is Model 753 from Campden Instruments, UK.

6. The capillaries are from Clark Electromedical Instruments (GC-100F 500 pcs).

7. If filament capillaries are not used, then the needles can be back-filled with a finely drawn out glass Pasteur. Use a rubber bulb with a small hole in the end. Cover the hole with your index figure to draw up or expel liquid into the pipet. (This is the same technique as used for filling Drummond Scientific microcaps.)

8. Alternatively, penicillin and streptomycin can be added at 20-50 |g/mL.

9. If left at 16°C the embryos will fail to gastrulate properly.

10. Three different Fast Red substrates have been compared: Vector™ Red alkaline phosphatase substrate (38,39), Vector Laboratories; Fast Red tablets, Boehringer Mannheim (40), Sigma Fast™ Fast Red TR/Naphthol AS-MX). The different Fast Red substrates have different characteristics. All are made up in Tris-HCl, pH 8.2. Vector Red is made up from three solutions provided in the kit from Vector Laboratories. Staining is quite rapid, appearing within a few minutes to 1-2 h. Prolonged incubation tends not to intensify the signal. The yolk stains yellow, but background in the embryo remains yellow. The Fast Red from Boehringer Mannheim is in the form of tablets. The solution throws down a precipitate after prolonged incubation. The signal develops less quickly than Vector™ Red, but produces a more intense red precipitate. Backgrounds can be quite orange in both the yolk and the embryo. Sigma Fast™ Fast Red is supplied as tablets for Tris buffer and the red substrate. It gives a similar result to Boehringer Fast™ Red in that the signal is intense and develops slowly. Backgrounds are a little less than with the Boehringer Fast™ Red, but greater than with Vector™ Red. It also produces less precipitate in the staining solution. The AP can be inactivated by heat treating at 65°C for 30 min. with the Vector™ Red precipitate but both the other precipitates are heat-labile and are lost if heat-treated. Thus, the AP activity must be inactivated by incubating in 100 mM glycine, pH 2.2, for 30 min.

11. It is important that the plasmid DNA is free of contaminants. Plasmid prepared by the various commercial resins is suitable (Magic™ or Wizard™ Minipreps Promega, Qiagen Plasmid Mini or Midi kit).

12. Treat with proteinase K to ensure that the DNA is free of RNase prior to riboprobe synthesis.

13. LiCl/ethanol precipitation does not completely remove unincorporated rNTPs, but this is not normally a problem.

14. In the case of fluorescein probes, the pellet is yellow, and unincorporated fluo-rescein-UTP runs at the front during electrophoresis and is easily seen on the UV-transilluminator. There should be a single, sharp band migrating behind the front. If it is smeared, then there is a problem with RNase contamination or with the RNA polymerase.

15. Previous protocols required that the probe should be partially hydrolyzed by heating in a sodium bicarbonate buffer (see ref. 42). This generates shorter lengths of probe, which more easily penetrate the tissue. However, this hydrolysis is difficult to control and tends to give lower signals. Now many laboratories omit this step. Perhaps with larger probes (>6 kb), partial hydrolysis may be worth considering.

16. Treat with proteinase K (10 |g/mL in PBT) to increase the permeability of the membrane. This is performed for 10-20 min at room temperature, depending on the stage of development. Embryos up to 24 h postfertilization do not need to be treated with proteinase K.

17. Use 1/100 of a standard digoxigenin or fluorescein riboprobe reaction in 200 |L hybridization solution as a starting point (equivalent to a final concentration of 0.5 |g/mL). Probes which give strong signals may be effective at 0.1 |g/mL.

18. This is a quarter of the dilution and longer incubation than recommended by Molecular Probes. However, this gives smaller crystals and less background. With zebrafish embryos, it is also not necessary to add 1.0 mM levamisole. Stain in 100-200 |L of staining solution in a 1.5-mL microfuge tube. Lay the tube almost horizontal, so that the embryos spread out and are equally exposed to the staining solution.

19. Note that other antifade mounting media may cause the ELF precipitate to fade.

20. Slight variations on this method are found in the following: Bayer and Campos-Ortega (10). Fix in 12.5% glutaraldehyde 10 min in 1X PBS, pH 7.2.Wash several times in PBT (0.3% Triton X-100, in 1X PBS, pH 7.2). Stain in 0.2% 5-bromo-4-chloro-3-indolyl-P-d-galactopyranoside in dimethylformamide, 5 mM K3FeIII (CN)6, 5mM K4Fe II (CN)6 in 0.1 M citrate buffer, pH 8.0, overnight at 37°C. Dehydrate embryos in ethanol, incubate for 5 min in xylene and mount in Durcupan. Prolonged incubation in xylene will remove the blue stain.

21. FDG is a substrate of P-gal. The compound is nonfluorescent, but on enzymatic cleavage, releases fluorescein. The embryos must be permeabilized before they can take up FDG. This is achieved by incorporating DMSO into the staining solution. The released fluorescein is freely diffusible, so the embryos must be examined immediately after staining.

22. The Imagene product was developed by Molecular Probes to overcome the problems associated with the necessity to permeabilize cells before they could take up FDG. The Imagene products are fatty acyl-modified FDG substrates. They are taken up without cellular permeabilization, and they yield fluorescent enzymatic hydrolysis products, which are retained in the cells. Once inside the cell, the substrate is cleaved by P-gal, producing a green fluorescent lipophilic product that is retained by the cells, probably by incorporation of the fatty acyl tail within the cellular membranes. Molecular Probes produce a variety of ImaGene kits with acyl-modified FDG of various chain lengths. The Imagene Green™ C12FDG lacZ Gene Expression kit includes a stock solution of C12FDG, chloro-quine (for reducing acid hydrolysis and endogenous P-gal activity) and phenylethylthio-P-d-galactopyranoside, which is a broad-spectrum galactosidase inhibitor for stopping the reaction. The Imagene Red™ C12RG lacZ Gene Expression kit (I-2906) contains a 12-carbon fatty acyl analog of the nonfluorescent lacZ substrate resorufin-P-d-galactopyranoside, which yields a red fluorescing enzymatic hydrolysis product.

23. A low-light video camera (SIT-57, General Electric) with an image processor (12) can be used or normal fluorescent optics.

24. DetectaGene™ Green lacZ substrate is an FDG analog that has been modified to react with intracellular glutathione. Once inside the cell, chloromethylfluorescein di-P-d-galactopyranosidase (CMFDG) is cleaved by intracellular P-gal, and then its chloromethyl moiety reacts with glutathione to form a tripeptide-fluorescein analogue. Since peptides do not readily cross the plasma membrane, the resulting fluorescent tripeptide cleavage product is much better retained than fluorescein. Although cells must be permeabilized to take up the DetectaGene™ Green substrate, its enzymatic cleavage products are brighter and more photostable than those of the ImaGene Green™ substrate; 200 pM CMFDG are as effective as 2 mM FDG. The fluorescent product stays in the cells for 3 d, whereas fluorescein diffuses out after 30 min. If cells are permeabilized by DMSO, in order for them to take up the substrate, they must undergo repair on returning to fish water, and presumably the enzymatic product does not diffuse out. DetectaGene™ Blue lacZ Expression kit contains 4-chloromethylcoumarin-P-d-galactopyranoside (CMCG), which forms a bright blue fluorescent membrane-impermeant product after reacting with intracellular glutathione. Visualization of CMCG fluorescent product requires a fluorescent microscope equipped with a DAPI filter set.

25. This method has been used to detect luciferase activity in embryos injected with a CMV-luciferase transgene construct. In preliminary experiments, Gibbs et al. (17) coinjected luciferin at 1 mg/mL with the DNA solution. However, injected embryos may also be incubated in a luciferin solution of 0.1-1 mg/mL. A lower concentration of 0.01 mg/mL detected only 10% as positives. Since F1 trans-genic fish failed to express luciferase, it has not been used successfully to iden tify stable transgenic lines. Homogenized embryos can be assayed for luciferase activity using a scintillation counter assay (41).

26. The modified plates are made by removing the outer rim with a hot knife and sanding the bottom of the plate with a belt sander until a hole appears in the bottom of each well. The hole is covered with a strip of Mylar sealing tape (Fisher Scientific No. 14-245-20). Embryos incubated in the plate are therefore close to the underlying film.

27. Orthochromatic film (TMAT-G) is used, since it is sensitive to 560 nm light, whereas normal X-ray film is not (XAR-OMAT). TMAT-H film gives similar results.

28. This requires a knifemaker suitable for making long-edge, extremely sharp "Ralph"-type disposable histological knives. Such knives can be used on a conventional microtome fitted with a suitable holder and will produce quality semithin sections on the order of 0.5 pm. A suitable knifemaker is available from Taab: K058 Taab Histoknifemaker complete with tools and glass.

29. PCR and Southern blotting are standard procedures, so they are not included here.

30. Taking fin biopsies followed by recovery of the fish requires a Home Office License in the UK. You should be familiar with your country's regulations for animal research.

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