Construction of Transgenic Zebrafish Protocol

Experiment Summary

In this experiment, EGFP-containing plasmid was introduced into zebrafish to observe its expression in animals. In zebrafish, green fluorescent protein could be observed under fluorescence microscope from proto-intestinal embryo to emergence stage.

Main Reagents

EGFP, green fluorescent protein gene, pEGFP-N2 vector, E. coli.

Main Equipment

Test tubes, test tube rack, adjustable microsparger, electrophoresis instrument, electrophoresis tank, staining tank, 42℃ constant temperature water bath, ice bath, constant temperature oscillation box, ultra-clean bench, manual microinjector, body microscope, borosilicate glass capillary, inverted microscope, camera system (CCD), automatic water circulation system set, incubation tank, culture dish, dissecting mirror, small-scale plasmid extraction kit.

Experimental Steps

1. Culture of transfected bacteria

(1) Preparation of medium: Weigh 3 g of tryptone, 1.5 g of yeast extract and 3 g of NaCl in a 500 mL triangular flask, add distilled water to 300 mL and dissolve. If prepare solid medium, add 4.5 g agar (1.5%), then adjust pH to 7.5 with NaOH. divide into 3 triangular flasks of 250 mL, cork and wrap.

(2) Sterilization and disinfection of culture medium: In a sterilization pot, add appropriate water, put in the wrapped culture medium, cover and heat. When the pressure rises to 0.05 MPa, open the vapor release valve to remove cold air. When the pressure returns to 0, close the vapor release valve, and when the pressure rises to 1.034 MPa hold for 15-30 minutes, stop heating, and when the pressure drops to 0, open the vapor release valve to remove the vapor.

(3) LB solid medium with Amp: Autoclave the prepared LB solid medium and cool it to 60°C. Add Amp (ampicillin) storage solution to make the final concentration of 50 μg/mL, shake well and spread the plate.

(4) Inoculate E. coli. containing plasmids separately with glycerol in a volume of 10 μL per tube.

(5) Incubate overnight at 37°C for 24 h in a shaker.

Extraction of plasmids in small amounts

(1) Sample processing: Collect 13 ml of the culture solution for 12-16 hours, centrifuge at 12000 rpm for 1 minute, and aspirate the supernatant as much as possible.

Note: If there is a large amount of bacterial fluid, the bacteria can be collected in 1 tube by repeated centrifugation. The amount of bacteria collected depends on the concentration of bacteria, too much bacteria collected will reduce the lysis effect.

(2) Suspension: Add 250 ul of solution I to the centrifuge tube where the bacteria have been precipitated, and then use a pipette to repeatedly blow or a vortex shaker to thoroughly suspend the bacterial cell precipitate.

(3) Lysis: Add 250 ul of solution II to the bacterial suspension, gently invert up and down 6-8 times to make the bacteria fully lysed, add 1 tube to mix 1 tube to ensure the solution is fully and timely mixed. The bacterial solution will become relatively viscous after sufficient lysis; to prevent DNA breakage, avoid violent mixing operation and lysis time too long.

(4) Neutralization: Add 350 ul of solutionⅢ, immediately reversing gently up and down 6-8 times, mix thoroughly, add 1 tube and mix 1 tube. At this point, a white flocculent precipitate will appear, stand for 2 minutes, centrifuge at 12000 rpm for 5 minutes (can be extended to 10 min).

Note: Solution III should be mixed immediately after addition to avoid local precipitation. If a small white precipitate is still present in the supernatant, the supernatant can be taken after repeated centrifugation.

(5) Adsorption: Carefully transfer the supernatant into the adsorption column, centrifuge at 12000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and then reinsert the column into the waste liquid collection tube. If the supernatant cannot be added at one time, repeat the operation (do not inhale the precipitate when transferring, this step is very important).

(6) Rinse: Add 600 ul of rinse solution PW to the adsorption column (check that anhydrous ethanol has been added before use) and centrifuge at 12000 rpm for 30-60 seconds.

(7) Repeat step 6 and discard the waste liquid in the collection tube.

(8) Place the adsorption column in the collection tube and centrifuge at 10,000 rpm for 2 minutes.

Note: This step should never be omitted to remove the residual rinse solution from the adsorption column, as the residual ethanol in the rinse solution will affect subsequent experiments (digestion, PCR, etc.).

(9) Elution: Place the column in a clean 1.5 ml centrifuge tube, add 50-100 ul of lysate TE or sterilized double-distilled water (pH 7.0-8.5) dropwise to the central part of the column membrane, let it stand at room temperature for 2-10 minutes, centrifuge at 12000 rpm for 2 minutes, then extract the plasmid DNA and store at -20°C.

3. The extracted plasmid was double digested, linearized and introduced into the fluorescent protein gene, and a band of 6.0 kb in size was obtained by 1.0% agarose gel electrophoresis, which was cut off and purified.

4. Zebrafish fertilized eggs were obtained by photoperiodic induction.

5. After the embryos developed to the protointestinal stage, the culture medium was gradually diluted with cold boiling water with violent gas, and after the embryos developed to the heartbeat stage, they were transferred to cold boiling water with violent gas and incubated in Holtfreter's. After the heartbeat stage, the embryos were transferred to the cold boiling water with storm gas.

6. Observe the expression of EGFP in transgenic zebrafish embryos under a fluorescent inverted microscope irradiated with 480 nm excitation light.

* For research use only. Not intended for any clinical use.

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Construction of Transgenic Zebrafish Protocol

Experiment Summary

Main Reagents

Main Equipment

Experimental Steps

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