Threespineトゲウオにおける遺伝子導入およびゲノム編集のためのマイクロインジェクション

The overall goal of this microinjection technique is to generate transgenic and genome edited stickleback fish to test gene and enhancer function. This method can help answer key questions in developmental and evolutionary genetics such as how genes and enhancers function to generate body pattern. The main advantage of this technique is that it efficiently generates transgenic fish for functional genetic analysis.

After preparing toll two plasmids and mRNA according to the text protocol, pull at least four micropipette needles from borosilicate glass. Store vertically in a capillary storage jar which the sharp ends facing down. To fertilize up to one hundred stickleback eggs, add 50 microliters of sperm solution and use a pipette tip to gently stir to ensure all eggs are fertilized.

While working on ice, back fill at least three needles by pipetting 0.5 microliters of injection solution into the blunt end of the needle and allow it to drain. In the meantime, turn on the transillumination light for the dissecting microscope and place a 13 square centimeter glass plate on the microscope light base with a 15 centimeter plaster saw blade on top of the glass plate. Orient the saw perpendicular to the injection apparatus with the indentations facing towards the needle holder then turn on the control box and ensure the pressure is set to approximately 175 kilopascals.

Set the injection duration to 180 milliseconds. Next loosen the needle holder, then insert a filled needle into the holder until resistance of the rubber holder can be felt and tighten the needle holder until it is finger tight. Then adjust the needle angle to approximately 45 degrees.

Use the micromanipulator controls to adjust the needle so that the end is centered in the field of view. Zoom in to approximately 40x magnification and focus on the tip of the needle, which should not be touching the glass below. With watchmaker's forceps grasp the tip of the needle two to three forceps'width from the end and gently break the tip at a 60 degree angle.

Then press the injection foot pedal several times to test whether the needle is broken. After a few taps, tiny red droplets should begin to come out of the end. Use a disposable transfer pipette to place a few drops of stickleback water on the glass plate.

Gently lower the needle into the water, then increase the back pressure until a faint stream of pink liquid emerges from the needle. Retract the needle as far as possible so that it will not be damaged while preparing the embryos. Approximately 25 minutes after fertilization, use two 10 microliter pipette tips to remove five to 10 embryos from the clutch and transfer to the glass plate.

With the pipette tips, gently separate the embryos into individual indentations of the saw blade using caution not the puncture the embryos. Next with a transfer pipette add enough stickleback water to cover the embryos. Then after three to five seconds, remove most of the water leaving only a small volume covering each embryo.

Starting with the embryo farthest away, slide the glass plate and zoom in on the embryo so that it fills approximately 25 percent of the field of vision. Lower the needle into the field of vision, then use a 10 microliter pipette tip to gently rotate the embryo to identify the blastomere, a grainy, slightly yellow, raised bump of cytoplasm on the top of the yolk. Rotate the embryo so that the blastomere is directly perpendicular to the end of the needle.

Lower the needle applying pressure slowly and evenly to pierce the corion and insert the needle into the cytoplasm without pushing it through the underlying yolk. Depress the foot pedal three to four times to inject so that a red bolus with slightly diffused edges fills approximately 1/8 the diameter of the cytoplasm. Having a sharp needle perpendicular to the corion will facilitate this step.

Limiting the amount of water added will help to keep the corion soft. And finally, targeting the cytoplasm will increase the likelihood of obtaining a transgenic animal. Use the micromanipulator controls to retract the needle, and with the 10 microliter pipette tip, hold down the embryo if it sticks to the needle.

After retracting the needle, slide the glass plate to align the next embryo with the needle and repeat the injection. When all embryos have been injected, use the transfer pipette with the end cut off to add water to the plate. Then use a transfer pipette to collect the embryos and place them in a 150 millimeter petri dish filled with stickleback water.

Incubate the embryos at 18 degrees Celsius. One day after injection gently pour off the stickleback water and replace with fresh water. Check for dead or malformed embryos and remove to prevent contamination.

As illustrated here, for transgenes that have enhancer activity, successful injection results in specific cellular expression of the transgene. For example, in the pectoral and median fins and the notochord. Injected fish can then be outcrossed to produce stable lines such as this back stable line that drives GFP expression in the embryonic heart at four days post fertilization.

For an active enhancer, 40 to 50 percent of embryos will show tissue specific transgene expression, for example, in the median and pectoral fins. Up to 100 percent of GFP positive F0 fish expressing toll two plasmid transgenes produce transgenic offspring. However, the percent of offspring carrying the transgene varies widely from less than one percent to 72 percent.

Saving only GFP positive injected embryos generally increases transmission efficiency. Only up to 10 percent of F0 back injected stickleback embryos show fluorescence in expected tissues. In addition, the transmission rate of backs is lower than that of plasmid constructs.

With only up to 14 percent of screened sticklebacks transmitting the back. In contrast to the low efficiency of back transgenesis, 70 to 100 percent of fish injected with talons have mosaic lesions. Here an uncut PCR amplicon in each of the injected embryos indicates that some cells in each embryo carry lesions at the target locus.

Once mastered, this technique can be done in a little over an hour for a clutch of 100 embryos if performed properly. While attempting this procedure, it's important to remember that practice and patience are required to improve technique and embryo survival. Following this procedure, other methods such as confocal microscopy can be used to answer further questions about detailed patterns of enhancer expression.

Also, genome edited fish can be grown in cross to study phenotypes resulting from induced mutations. After it's development, this technique paved the way for researchers in the field of evolutionary and developmental genetics to study gene regulation and function in sticklebacks. After watching this video, you should have a good understanding of how to inject stickleback embryos for transgenesis and genome editing.