Transgenesis in drosophila is essential for studying gene function at the organism level. Embryo microinjection is a crucial step for construction of transgenic flies. Here, we take the phiC31 integrase-mediated transgenesis in drosophila as an example, and present a detailed protocol for embryo microinjection for transgenesis in drosophila.
Injection needles whose tips beveled by a grinder to mask embryonic block, or less large cytoplasmic leakage. However, the grinding solution can enter the beveled tip of a needle during the grinding process as the grinding solution inside the tip does not naturally dry up quickly. Our protocol uses a foot air pump with a pressure gauge to apply air pressure to a needle while grinding its tip.
This prevents the grinding solution from siphoning, thereby the grinding solution is prevented from entering the beveled tip, enabling the built needle to be used in method. To begin, wet a cloth with grinding solution. Fix the cloth on the needle grinder, then connect a needle to a foot air pump using a silicone capillary tube.
Mount the needle onto the needle holder of the grinder. Adjust the angle of the needle, such that it is bent 35 degrees to the grinding plate. Use the coarse control knob under the microscope to adjust the needle tip's position until it is placed just above the grinding surface.
Next, use the foot air pump to keep the inner pressure of the needle at 40 pounds per square inch, then use the fine control knob to make the needle tip touch the grinding plate. After grinding for three to five seconds, unload the needle when air bubbles start to exit the tip. Next, heat a 200 microliter pipette with the outer flame of an alcohol burner.
When the pipette tip starts to melt, stretch it out. Cut off the sealed end of the pipette tip with scissors. To dechlorinate drosophila embryos, remove the flies from grape juice agar plates with a brush.
Rinse the collected embryos in a cell strainer with double distilled water. Dry the cell strainer with tissue paper, then place the dried strainer in a Petri dish containing 30%bleach. Manually stir the cell strainer to ensure even mixing.
Rinse the embryos in double distilled water for two minutes before picking out the floating embryos with a paintbrush to make a lineup for further transgenesis. To begin, with a paintbrush, transfer dechlorinated drosophila embryos onto a long strip of grape agar. Use a dissecting needle to line about 60 embryos along the edge of the strip, with their posteriors facing outwards.
Now place an 18 millimeter long double-sided tape lengthwise along the edge of a cover slip. After removing the backing, gently press the cover slip onto the aligned embryos. Transfer the cover slip with embryos into a box containing a desiccant.
Add halocarbon oil over them. To microinject the embryos, first load two microliters of DNA into a needle using a stretched out pipette. Place the aligned embryos with halocarbon oil under an inverted microscope.
Next, use a micromanipulator to insert the needle into the posterior ends of an embryo. With the foot control, carefully inject the DNA into the embryo. Remove the needle after completion of microinjection of the first embryo, then move it to the posterior end of the next embryo.
Transfer the injected embryos into an agar plate for incubation. When the eggs have hatched, prepare a vial with food. Add a few drops of distilled water to the vial.
With a stereo microscope and a paintbrush, collect the hatched larvae, then transfer them into the vial. Incubate the vial at 25 degrees Celsius under 50 to 60%humidity for 10 days. Over-dried embryos appeared deformed and cannot be used for microinjection.
Under-dried embryos leaked a large amount of cytoplasm when punctured, thereby reducing survival rates. Appropriately dried embryos leaked only a small volume, if any, when injected. The drosophila line expressing the phiC31 integrase used for embryo microinjection had white eyes.
The aged flies of this genotype have pink eyes resulting from accumulated red fluorescent protein expressed specifically in the eye. Transgenic flies bearing a transgene at the ATTP2 docking site with orange eyes were successfully produced.
