The overall goal of this procedure is to deliver nucleic acids into the embryos of Ceratitis Capitata, an important agricultural pest, to ultimately expand the toolbox for its control. This method helps functionally correctorize genes in an immobil organism, as well as generate transgenic strains. The main advantage of this technique is that very low amount of nucleic acids are injected to achieve the desired effects.
Maintain all the life-stages of the medfly colony at 25 degrees Celsius with 65%humidity and a 12 12-hour light/dark photo-period with a photo-phase starting at 8 a.m. Insert a sponge strip through a small opening in the cage's base to provide the flies with water by means of capillary action. You establish a six liter cage, load one with between 1, 500 and 2, 000 pupae.
For food, load the cage with about 45 grams of a one to 10 mixture of yeast and sugar. To collect eggs, place a plate filled with water below the cage. Females will deposit eggs through the mesh, which will then collect in the water.
Because of virgin females can ova-posit, ensure that the eggs are all fertilized by waiting until the flies are at least a week old, before collecting eggs. To collect eggs, filter the water with a fine-strainer. Then using a pasteural pipette, transfer the eggs into a plastic box loaded with about 0.5 liters of standard larval food.
If not enough eggs were laid, then increase the yeast content of the adult's food up to 25%Place each box in a transparent container, containing a layer of bran to favor pupation. Cover the container with a net lid to allow air circulation. After about 10 days, check for third-instar larvae that have jumped down into the bran layer for pupation.
Once found, prepare to check for pupae on the following days. To collect the pupa, use a soft brush to sweep them up from the container. Initially, collect the pupae in a small cup.
After the collections are completed, transfer the pupae to a new adult cage. The adults should emerge in about 10 days. For the micro-injection, have slides prepared in advance.
Attach a two centimeter strip of double-coated tape to a slide and mark the edges with a china marker to make a wall for an oil layer that keeps the embryos hydrated. To collect freshly laid embryos, allow young fertilized females to ova-posit for 30 minutes on a new tray and then collect the embryos. To remove the chorion from the embryos, dip the strainer and eggs into a commercial 50%bleach solution for five seconds.
It is critical to not over bleach the embryos. Immediately wash the bleached embryos by carefully dipping the strainer into clean ultra-pure water. Do this at least four times.
Then, keep the strainer in clear ultra-pure water and inject the embryos within two hours. Next, using a fine paintbrush, collect about 50 embryos and place them on a disc of black filter paper soaked with water. Under a dissecting stereo microscope, row the embryos onto the double-coated tape of a prepared microscope slide.
Orient all the embryos in the same direction. It is important to manipulate the embryos carefully because the piercing of the embryo membrane with the paint brush may cause damage that reduce the embryo's survival. Now cover the embryos with a layer of chloro-trifloro etholene oil.
The oil should be contained by the china marker borders. Don't overflow the borders. Using a micropipette, load the injection needle with a few microliters of nucleic acid solution at one to two micrograms per microliter.
Then, connect the needle to the micro-injection apparatus and set it to provide a half second injection at 800 hecto-pascals with a back pressure of 150 hecto-pascals. To inject, use a foot-pedal trigger and use the micro-manipulator to control the needle while using the stage controls to move the slide. Confirm that the injection bolus is being ejected properly and then align the needle with the posterior pole of the first embryo in the row.
By moving the stage, insert the needle into the embryo and inject the bolus of nucleic acids. The injected liquid will induce an increase in internal pressure resulting in a slight movement of the embryo. Release the pedal, and immediately but gently, remove the needle from the embryo.
A little cytoplasm will leak at the injection site. This is normal. If embryos are bursting with the injection, then let the embryos desiccate for a few minutes before covering them in oil.
Following the injection, transfer the slide to a petri dish. Place a disc of filter paper in the lid of the dish. Soak the filter with water and close the petri dish with paraffin film.
Incubate the chamber at 25 degrees Celsius. Two days after the injection, check the slides several times throughout the day for hatched larvae. Look for larvae in the oil and using a fine paintbrush, transfer them to a petri dish with carrot-based larval food.
Load each dish with up to 200 larvae. The larva cannot survive in the oil long, so search often. Incubate the larvae with the lid mostly closed, so there is still air-flow to the larvae.
Check the dishes daily and after a week prepare to collect larva by placing the dishes in a container with bran. Collect the pupae and soon after expect them to emerge. Later anesthetize hatched adults using carbon dioxide and inspect them for transformance.
When screening out, that's mobilization method can be critical. Medfly adults can be anesthetized using ice or CO2 but prolonged exposure might reduce other's survival. Check for the presence of red fluorescence using a DS red wide filter.
Or check for green fluorescence using a EYFP filter. Transfer the transformed individuals into a small cage with wild-type adults of the opposite sex to establish a heterozygous strain. In one experiment, double-strained RNA was used to silence inx5 gene expression.
The adult germ-line of uninjected controls appears to be normal as expected, while treated individuals showed underdeveloped testes and ovaries. Quantification of inx5 transcript abundance in the abdomens of individuals from both sexes confirmed the significantly lower expression of the gene as compared to the controls. In a second experiment, two strains with labeled sperm were generated using the beta-2 tubulin promoter to drive different fluorescent markers.
Transgenic males showed a distinct fluorescence pattern in the body and in the testes. With this video, we provide our optimized protocols for the rearing and micro-injection of nucleic acids into medfly embryos. Once mastered, up to 1, 000 embryos can be injected over an eight hour working day.
Following this procedure, other metals like thy-lino-crisp-er, cas-min-eta-geno-meta-tine can be performed to develop a new generation of medfly strains.
