TY  - GEN
T1  - Serum-free adapted Drosophila S2R+ line is amenable to RNA interference
AU  - Luhur, Arthur
AU  - Mariyappa, Daniel
AU  - Klueg, Kristin M
AU  - Rogers, Stephen L
AU  - Zelhof, Andrew C
DO  - 10.17912/micropub.biology.000362
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000362/
AB  - Drosophila Schneider S2 cell lines are susceptible to RNA interference (RNAi) and this attribute has cemented Drosophila cell lines as an important tool for high throughput functional genomics screening (Rogers and Rogers 2008; Zhou et al. 2013; Mohr 2014). RNAi against Drosophila Rho1 in S2 cells results in a block in mitosis, giving rise to enlarged and multinucleated cells (Rogers et al. 2004). Recently, we have adapted a select group of Drosophila embryonic cell lines to grow in media supplemented by adult fly extract (FEx), instead of fetal bovine serum (FBS) (Luhur et al. 2020). Here, we demonstrate that S2R+ (FEx 2.5%), the M3 + 2.5% FEx-adapted S2R+ line is also amenable to RNA interference (RNAi), similar to its parental S2R+ cells cultured in M3 BPYE + 10% FBS. We observed similar efficacious RNAi against Rho1 in S2R+ and S2R+ (FEx 2.5%) as the cells became enlarged (Figure 1A-D), multinucleated (Figure 1E-H) and failed to proliferate (Figure 1I). There was a comparable growth delay in S2R+ and S2R+ (FEx 2.5%) cells treated with Rho1 dsRNA, as the cell population doubled in 7 days (Figure 1I). In contrast, both S2R+ and S2R+ (FEx 2.5%) cells treated with double stranded RNA against a control target gene encoding the bacterial antibiotic resistance gene chloramphenicol acetyl transferase (cat) had significantly proliferated 5 fold more under similar conditions (Figure 1I). In addition, there were no significant differences in the growth ratio between S2R+ and S2R+ (FEx 2.5%) (Figure 1I) (Luhur et al. 2020). These results demonstrate that Rho1 RNAi was recapitulated robustly in S2R+ (FEx 2.5%), similar to its parental S2R+ cells. Lastly, to confirm the depletion of Rho1, we assayed for Rho1 protein levels in these cultures by Western blot. Our result indicated a strong reduction in the amount of Rho1 protein in both S2R+ (FEx 2.5%) and S2R+ cells after Rho1 knockdown (Figure 1J). In contrast, the control RNAi knockdown of cat did not affect Rho1 protein levels in either S2R+ or S2R+ (FEx 2.5%) (Figure 1J). In summary, this finding expands the utility of the fly extract-adapted cells for their use in functional genomics in a more physiologically relevant culture condition.
PY  - 2021
JO  - microPublication Biology
ER  -