This product is optimized to level proteins with the fluorescence protein tag, and to visualize the location and the expression of the protein of interesting zebrafish lab individual. It is especially useful for imaging proteins expressed at a low abundance. Our method simplifies and accelerates the donor construction process.
These components, except for the CDS, are incorporated into the backbone beforehand. The donor was also optimized to increase the efficiency. The transmission screen workflow accumulates inheritable nucleus at the lowest cost of time and effort.
Zebrafish is the model organism widely used in research on infection and immunity. We are especially interested in the mechanisms underlying sepsis. Since we are now able to do fluorescence tagging easily using our protocol, we will try to label an image, sepsis proteins and cells in zebrafish.
To begin, place one healthy female zebrafish and two healthy male zebrafish into a mating tank. Use a divider to separate the males from the females to prepare them for mating. On the day of microinjection, prepare the microinjection mix on ice under an RNase-free environment.
Using an Rnase-free pipette tip, load the micro injection solution into the needle. Place the loaded needle into a micro manipulator attached to a micro injector. Under a microscope, use pointed tweezers to cut the tip of the needle by gently pinching it until it breaks.
Adjust the injection pressure until the needle consistently ejects a one nanoliter droplet. Next, remove the divider from the mating tank, and allow the fish to breed for six minutes. Collect and transfer the one cell stage embryos into a Petri dish containing embryo buffer.
Examine the health of the embryos under a light microscope and remove unfertilized eggs or debris. Set aside 20 healthy embryos in a separate Petri dish as uninjected controls, and label the dish. Using a small brush, transfer and gently line up healthy one cell stage embryos onto the grooves of a microinjection plate warmed to room temperature.
Remove excess buffer from the plate. With the help of a needle tip, gently adjust the position of each embryo so that the animal pole faces the needle. Inject one nanoliter of the mixed solution into the animal pole.
After injection, gently rinse the embryos into a 6-well plate with embryo buffer. Place the plate into a 28.5 degree Celsius constant temperature incubator. At 10 hours post fertilization, examine embryonic development under a stereo microscope and remove dead embryos.
At 24 hours post fertilization, collect embryos into 1.5 milliliter tubes for genotyping, and discard extra water carefully. Add 100 microliters of lysis buffer containing Proteinase K to each tube. Incubate the tubes at 56 degrees Celsius for one hour, and then at 95 degrees Celsius for 15 minutes.
After incubation, add 200 microliters of ethanol and mix well. Centrifuge the tube at 19, 000 G for 10 minutes and discard the supernatant before resuspending the pellet with 500 microliters of 70%ethanol. Centrifuge again and air dry the pellet before dissolving it in 50 microliters of double distilled water.
Use one microliter of the extract to prepare the reaction mix for the PCR. After PCR, run agarose gels with two microliters of PCR products to confirm successful amplification. Perform enzymatic digestion to evaluate the efficiency of the sgRNAs.
Gently mix the digestion solution and incubated at room temperature, following the manufacturer's instructions. Run agarose gels for digested products alongside undigested PCR controls to confirm digestion. To analyze sgRNA efficiency, open the TIDE website and click Start TIDE.
Input the guide sequence of the sgRNA into the guide sequence frame. Click Browse to upload the sequencing result of the wild type control into the control sample chromatogram field. Similarly, upload the sequencing result of the edited sample into the test sample chromatogram field.
Click Update View to analyze the results. To begin, use ligation independent cloning or LIC with exonuclease III to construct the donor plasmid. Design primer F1 with an overlapping sequence with the vector element 1 and part of element 2.
Design primer R2 with a part of element 1, element 2, and element 3. Design primer F2 to include elements 3 and 4, and the 5 prime sequence of the CDS following the sgRNA target site. Design primer R1 with the three prime sequence of the coating sequence and an overlapping sequence with the vector.
Use zebrafish CDNA as a template and perform PCR with the designed primers. Purify the PCR products using ethanol precipitation. Digest the donor vector with restriction enzymes such as pCI and Acc65I, and purify the digested donor vector.
Quantify the purified PCR fragments and digested donor vector using DNA gel electrophoresis. Next, prepare the LIC mix with purified donor vector and incubate on ice for 60 minutes. Add one microliter of 0.5 molar EDTA to stop the reaction.
Pipette up and down to mix the solution. Incubate the reaction mix at 60 degrees Celsius for five minutes. Cool the LIC products on ice.
Then, transform DH5-Alpha competent cells using LIC products. Plate the transformed cells on Luria-Bertani plates containing the ampicillin, and grow them for 16 hours. After incubation, pick single colonies from the LIC plates for enzymatic digestion analysis and DNA sequencing.
Extract and purify correct donor plasmids with a purification kit. Inject zebrafish embryos with a microinjection mix for gene knockin'12 to 48 hours after microinjection, under a stereo fluorescence microscope, observe the fluorescence of the zebrafish larvae. Using an established screening method, select the fluorescent larvae and rear them separately.
At one month of age, collect a small piece of the coddle fin from each anesthetized zebrafish into a labeled PCR tube. Place the corresponding anesthetized zebrafish in a 6-well plate filled with UV sterilized filtered water matching the label on the tube. Extract genomic DNA using the alkaline lysis method.
Design a forward primer upstream of the left homology arm and a reverse primer on the insert. Perform PCR, targeting the five prime junction of the sequence. Examine the F1 embryos obtained by mating the screened F zebrafish with wild type zebrafish for GFP expression patterns.
Genotype the F1 embryos using junction PCR. Inheritable specific fluorescence was observed in muscles of all F1 progeny, indicating the successful GFP labeling of connexon 39.9. Inheritable specific fluorescence was detected in the lens of all F2 progeny, suggesting successful mCherry tagging of connexon 44.1.
Correct knockin'of connexons 39.9 and 44.1 was verified through junction polymerase chain reactions.
