When preparing to transform Drosophila embryos, several days of preparation are required before injections begin. Flies that are deficient in the allele that is to mark the transformants, most often ry506 or w1118, must be in abundant supply, both for egg collections and later crosses. Bottles of flies for egg collection are set up by taping an egg laying plate to the bottom of a plastic beaker with air holes punched in it with a 20 guage or smaller needle. Bottles should be moved to a day for night schedule at least two days before collections begin, as this will improve the number of eggs being laid.
Prepare the injection solution with a 3:1 molar ratio of insert to helper plasmid, with a total concentration of 0.6 mg/ml. DNA used for injections should be made by Quiagen or CsCl prep. Since such small amounts of solution are used in the injection process, a 20 ul final volume should be sufficient. After mixing the DNA in its proper amounts, ethanol precipitate, spin and wash with cold 70% ethanol. Allow pellet to dry and dissolve in injection buffer solution (0.1mM Na Phosphate pH 7.8, 5mM KCl). Just before filling the needle, spin injection solution for 10 minutes to avoid any particulate matter that might enter and later jam it.
Preparing the needle
There are a variety of ways to prepare needles. This is one of the most important steps for injections, as the sharpness of the needle and its ability to stay unclogged will to a large degree determine how many embryos survive the process and thus how many days will have to be spent injecting. I use a method of beveling that uses a slurry of grinding powder and a regular magnetic stirring set-up (see technical notes by James Powers, DIN Vol. 12) The slurry is made from silicon carbide powder (Grit 120; Buehler Ltd., Lake Bluff, IL) and ddH2O at a 1:3 ratio. The grit must be washed several times to remove the smallest particles which will remain suspended after the bulk has settled out. The slurry should be autoclaved before use and every week to prevent any bacterial contamination. A shallow beaker should be used. For injections we use a 1mm capillary pipet, pulled to form a needle and then back filled with the DNA solution before sharpening. With the slurry being stirred at a relatively fast speed, but not so fast as to throw grit everywhere, the tip of the pipet should be inserted at 135û angle with respect to the direction of flow of the slurry. By holding the needle steady for 4-5 minutes, the tip of the needle will be beveled to a sharp point. This may provide a wide enough opening for injecting, but if the DNA does not flow freely from the tip when injecting begins, gently touch the tip of the needle to the edge of a slide under a microscope while applying gentle positive pressure with your syringe. One side of the tip will be weakened from the beveling and should break easily, providing a wide but sharp tip for injections.
Eggs must be collected every hour and injected within the hour, in order to introduce the DNA before cellularization takes place. Before the first round of injections, a fresh egg laying plate with yeast paste should be put on the bottle for half an hour to induce the flies to lay any over developed eggs they may have been carrying. The eggs are then collected every hour by exchanging a fresh plate with yeast paste for the old plate, which is then wetted slightly and brushed lightly to release the eggs from the agar. The eggs are then strained out and washed in ddH2O or PBS.
The chorion, the "shell" on the embryos, must be removed before injecting. Although some use bleach for this step, this drastically reduces survival rates. We manually dechorionate by brushing the embryos onto a piece of double-stick tape. A relatively sharp tipped instrument (a push-pin stuck on a pencil will do) is used to gently rub the side of each embryo till it rolls over and pops out of its chorion. The embryo is then picked up and placed on the edge of a thin strip of agar cut from an egg laying plate. The eggs should all face in the same direction, with their anterior end facing out from the agar. You can recognize the anterior end by the dorsal appendages, which are two string like parts that come off the egg with the chorion. Also located on the anterior end is the micropile, a small nipple-like extension from the egg that should be visable after removing the chorion. When 40-50 embryos have been lined up on the agar strip, a coverslip with double stick tape on it is gently lowered onto the eggs. The eggs should end with one quarter to one fifth of their posterior ends over the edge of the double stick tape. It may be a good idea to use 1/4" 3M, type 415 tape, as this tape has supposedly been tested non-toxic, whereas other double-stick tape has not (see technical notes by Mary Whiteley and Judith A. Kassis, DIN Vol. 11) The coverslip should then be placed in a container with Drierite for 5-15 minutes. This time should be adjusted until the embryos are loose enough to inject without leaking, but not so loose as to appear "wrinkled" or bag-like. After desiccation, the eggs are covered by a thin layer of oil. We use Halocarbon Oil Series 700, CAS# 9002-83-9, Halocarbon Products Corporation.
This is obviously the point at which most of the embryos get killed. The needle should be inserted quickly in the center of the posterior end and the injection should place as much of the solution as close to that end as possible, as this is where the germ cells we want to transform are located. The needle should then be pulled out quickly to avoid any leakage. There are several problems that will tend to arise during injections, each with its own solution.
- If embryos leak immediately upon being punctured, they have been under-desiccated. Do the best you can with this slide and increase the desiccation time by a few minutes next time.
- If many of the embryos are "empty shells", reduce the desiccation time.
- If the eggs are not easily punctured by the needle and leak often when the needle is pulled out, the needle is not sharp enough. Try re-breaking the tip on the edge of the slide, or make a new needle.
- If the embryo leaks while you are injecting, you are trying to inject too much DNA solution. Use a lighter touch or a smaller syringe.
- If the needle jams frequently, there is either an excess of particulate matter in the injection solution or the needle's opening is too small. Remember to spin down the solution before back-filling your needle.
Eggs should be placed in a humidified environment at 18ûC. They should be checked every twelve hours in order to verify that they are properly humidified and covered with oil. Add as much oil as is nescesary to keep the eggs covered, although not too much. Larva will begin hatching after two days and should be collected at least twice a day using the same type of instrument that was used for dechorionation. Do not collect larva until they have freed themselves from their egg shell. Place around 50 larva in each vial of fly food (10ml of fly food). Water food regularly or keep well humidified.
Collecting G0s and transformants
After hatching, G0 flies must be collected before mating. Approximately half of these flies will be sterile, due to damage of the developing sex organs done by injecting. Cross G0s with the original marker stock. Place two G0s of the same sex in a vial with six marker flies (opposite sex, obviously). Screen their progeny (G1) for transformants with the marker gene. Take only one transformant from each vial, as transformants from the same vial are likely to have the identical insertion site.