解剖、イメージング、および薬理学的治療： ショウジョウバエ蛹精巣や独身男性生殖ライン嚢胞のex vivo培養

The overall goal of this procedure is to prepare ex vivo cultures of drosophila, pupil testes and germline cyst to perform live imaging studies and inhibitor treatments to analyze post miotic For myogenesis, this is accomplished by first dissecting intact testes from early puy 24 hours after purium formation. The second step is to dissect single germline cysts from these testes, then intact testes and single germline cysts can be used for live imaging experiments. Alternatively, the next step is to incubate the intact testes or germline cyst with inhibitor compounds.

Ultimately, immuno staining of squash, testes and fluorescence microscopy are used to monitor the effect of the treatment. The main advantage of this ex vivo culture technique over existing genetic methods is that it allows access to dilo spermatogenesis during post miotic stages. In general, individuals new to this method with struggle because some experiences needed to dissect and handle pupil tissues.

To begin the experiment, distribute drops of 80 microliters of shields and San M three insect medium with fetal bovine serum and antibiotics into a 10 centimeter petri dish. Next, using a broad pair of forceps, pick a pupa and transfer it to a drop of medium on the dish. Then transfer the materials to a stereo microscope equipped with a fiber optic illuminator.

Take care not to heat the testes above room temperature during the dissection using number five forceps. Grab the pupa at its most posterior end and hold it in position. Grab the anterior sphericals with another pair of forceps and open the pupil a perm at its anterior end.

Peel off the pupil case until at least part of the thorax is exposed. Continue to hold the pupa in position by its most posterior end, and release the internal pressure of the pupa by inserting both arms of a pair of forceps into the head region of the pupa. Next, rip the pupa open by opening the forceps and moving the forceps in the anterior direction and ensure that the opening is as large as possible in the first attempt.

Avoid damaging the pupa at its posterior end before the pressure has been released. As this might result in the testes being pushed directly through the small opening and damaged. Once the pupa is opened, the released internal pressure of the pupa presses out glomerate of fat body cells and tissue through the large opening.

Then increase the size of the opening using one pair of forceps and avoids squeezing the pupa with the forceps as this might damage the testes. Next, hold the pupa at its most posterior end close. The other pair of forceps and gently streak out the contents of the pupa from posterior to anterior to release the testes from the pupa.

Check whether the pupil testes have been pushed out. They will not be connected to any other tissue. At 24 hours A PF collect the testes with a precut 200 microliter pipette tip.

Transfer only a small amount of the medium to reduce the number of fat body cells carried with the testes. Then place the testes into medium in a fresh culture dish. Wash the testes several times with medium until only few fat body cells remain for live imaging.

Transfer testes to a glass bottom culture dish with medium and use an inverted imaging microscope to begin dissection. Transfer one or more pupil testes to a glass bottom culture dish filled with medium. Use a stereo microscope with fiber optic illumination and two stainless steel needles for the dissection of male germline cysts.

With one needle carefully pinned down, one testis at its anterior or posterior end and hold it in position. Insert the other needle into the testis and disrupt the testis sheath by moving the needle sideways, which will release many of the cysts. Continue to hold a testis in position with one needle.

Then use the other needle to gently streak out the remaining cysts from the testis. Next separate aggregated cysts. Using a 200 microliter pipette tip by transferring the cysts to a fresh cultured dish with medium.

Then move the cultured dish to an inverted imaging microscope for live imaging of single cysts. Disperse the cysts again with a needle if necessary, identify the stage of cysts by using phase contrast and fluorescence microscopy. Focus on a cyst of the desired stage.

Confirm the focus and the position of the cyst frequently. During longer imaging experiments, the cysts do not adhere to the bottom of the culture dish. Intend to float out of focus.

Fill each well of a 24 well cell culture plate with 500 microliters of medium for one experiment of 12 replicates. Then transfer one pupil testis to each well using a precut 200 microliter pipette tip. Next check for the presence of protamine in the isolated testes using an inverted fluorescence microscope.

The testes should be free of protamine, B-E-G-F-P signal, which indicates that the hisone to protamine or HP switch has not taken place in any of the cysts. Replace testes that already show positive cysts. Add 500 microliters of medium containing endo caric acid, the inhibiting compound to reach a final concentration of 150 micromolar in one milliliter to each of the first 12 wells.

Use the remaining wells as untreated controls by adding 500 microliters of medium with solvent. Then incubate the plate at five degrees Celsius for 24 hours in the dark. Check again for the presence of protamine in the incubated testes.

The control testes should now show one or more protamine, B-E-G-F-P positive cysts, which indicates a transition through the HP switch. Next, using a pipette with a 200 microliter tip, transfer the testes to poly L lysine coated slides. Perform squash preparations and stain with fluorescent antibodies.

Following published protocols, phase contrast images of drosophila testes were obtained using this dissection method. This is a slightly squashed whole mount testes of a drosophila pupa. At 24 hours after PY formation or a PF spermatogonia are restricted to the anterior tip beneath the hub region.

The remaining testis is filled with spermatocytes spermatids in the Nevin current stage with round nuclei and spermatids in elongating stages with growing flagella overlays of phase contrast, EGFP and RFP fluorescent images from home mount pupil testes were obtained to detect H two A-V-D-R-F-P and protamine B-E-G-F-P at 24 hours A PF.None of the spermatids have undergone the HP switch frequently. The testes dissected at this stage contain an autofluorescence structure. A pupil testis dissected 36 hours A PF displays the first protamine, B-E-G-F-P positive cyst.

Pupil testes 48 hours A PF have several germline cysts with visible protamine positive nuclei. Time-lapse ex vivo live images of whole testes developing in culture can be recorded. Using this method, A pupil testis was dissected 45 hours A PF incubated in medium for six hours and imaged every five minutes.

Within this timeframe, several cysts of spermatids emerge from the HP switch stage and show a bright protamine B-E-G-F-P signal. After watching this video, you should have a good understanding of how to obtain early pupil testis and single germline cyst in culture in order to perform in vivo imaging or inhibitor treatments.