To silence each candidate gene in the female germline cells, three males from the respective RNAi stock were crossed with three virgin females from the nos-GAL4-VP16 MVD1 driver line. Fly food used in crosses was supplemented with some dry yeast. The vials were incubated for 7 days at 25 °C before removing the parents. Ten days after crossing, non-balanced female progeny was selected for maturation and dissection. To stimulate oogenesis, five females were incubated in the presence of three males and dry yeast on fly food for 2–3 days at 25 °C prior to dissection.
The workflow for each candidate gene comprised four steps: (1) RNAi-mediated knockdown of the candidate gene in ovaries, (2) stimulation of oogenesis in females, (3) dissection of ovaries and DNA staining, and (4) examination of the karyosome using confocal microscopy. The ovary size of females was noted during dissection and classified as either normal, small, or no/tiny in comparison to wild-type females. In the initial round of the screen, the karyosome was examined in about six oocytes between oogenesis stage 3 and stage 9 per candidate gene. The karyosome could not be examined when the candidate gene knockdown resulted in tiny or completely underdeveloped ovaries. The karyosome was classified as “normal” when its shape was spherical, slightly deformed, or slightly elongated. The karyosome was classified as “abnormal” when the chromatin formed a strongly distorted mass or discontinuous chromatin masses. To assess how candidate gene knockdown affects karyosome morphology, both the frequency and severity of observed karyosome defects were considered. All candidate genes with dissectible ovaries were classified based on karyosome morphology. The genes were considered to have “abnormal” karyosomes, when at least three out of six examined oocytes showed abnormal karyosome morphology. In addition, when one or two oocytes showed an abnormal karyosome morphology, the genes may be considered to have “abnormal” karyosomes depending on the overall impression across the karyosomes. These genes with “abnormal” karyosomes were selected for the second round of the screen for validation of the karyosome defects.
The workflow for each candidate gene comprised four steps: (1) RNAi-mediated knockdown of the candidate gene in ovaries, (2) stimulation of oogenesis in females, (3) dissection of ovaries and DNA staining, and (4) examination of the karyosome using confocal microscopy. The ovary size of females was noted during dissection and classified as either normal, small, or no/tiny in comparison to wild-type females. In the initial round of the screen, the karyosome was examined in about six oocytes between oogenesis stage 3 and stage 9 per candidate gene. The karyosome could not be examined when the candidate gene knockdown resulted in tiny or completely underdeveloped ovaries. The karyosome was classified as “normal” when its shape was spherical, slightly deformed, or slightly elongated. The karyosome was classified as “abnormal” when the chromatin formed a strongly distorted mass or discontinuous chromatin masses. To assess how candidate gene knockdown affects karyosome morphology, both the frequency and severity of observed karyosome defects were considered. All candidate genes with dissectible ovaries were classified based on karyosome morphology. The genes were considered to have “abnormal” karyosomes, when at least three out of six examined oocytes showed abnormal karyosome morphology. In addition, when one or two oocytes showed an abnormal karyosome morphology, the genes may be considered to have “abnormal” karyosomes depending on the overall impression across the karyosomes. These genes with “abnormal” karyosomes were selected for the second round of the screen for validation of the karyosome defects.
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