Isolamento de Drosophila melanogaster Testes

The overall goal of this procedure is to isolate testes from zero to three day old male drosophila melan gaster. This is accomplished by anesthetizing male flies and dissecting out their testes in a drop of ice. Cold PBS under a microscope using two dissecting needles, pull the fly's abdomen away from its thorax.

Finally tease the testes from the rest of the tissue and collect them for use in a variety of analytical techniques such as immunofluorescent, staining, or total RNA and protein isolation. Visual demonstration of this method is critical as the identification of the dissected testes can be difficult to learn cause they're hard to distinguish from the gut. Demonstrating the procedure will be shemei ma, a technician from my laboratory.

First assemble a bucket of ice, dissecting needles and about 100 milliliters of phosphate buffered saline. Place the PBS in the ice bucket to chill. Make a microscope slide with a black background by cutting white 20 pound paper to fit the slide.

And then color one side black. Using a permanent marker, glue the paper in place on the slide and secure the slide to the CO2 pad. Under a dissecting microscope at the dissecting microscope, anesthetize a bottle of zero to three day old flies with a stream of CO2 and empty them onto the pad.

Near the affixed microscope slide, identify male flies by their characteristic dark colored mating hooks and sort out between 50 to 100 pipette about 50 microliters of ice cold PBS onto the center of the microscope. Slide forming a drop. Pick up a male fly by dipping a dissecting needle into the drop of PBS and gently touching the fly's ventral side, causing it to stick to the needle.

Place the fly in the drop of PBS firmly press one needle between the thorax and the abdomen of the fly. Press the other needle between the sixth and seventh abdominal segments, and then pull the abdomen away from the thorax. Tease the testes apart from the rest of the organs, especially the gut, which can look similar to help differentiate the testes in the gut.

Remember that testes always come in, pairs have a characteristic spiral shape and are more white than gut tissue. Collect the testes in a micro centrifuge tube containing ice cold PBS and store on ice where they can be stored for several hours. Once mustard, one fly can be dissected in less one minute following this procedure.

Other methods, like deep sequencing of small silencing RNAs or microRNAs can be performed in order to answer additional questions, such as how does the repertoire of RNAs change in a particular mutant fly?