Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes

Abstract

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

Genome-wide activities of Polycomb complexes control pervasive transcription

“…To study the effects of pervasive H3K27 methylation, we used cultured cell lines carrying homozygous mutations in PcG genes, created by the procedure of Simcox et al (2008) (see Methods). Line EZ2-2 is homozygous for E(z)…”

Hippo signalling in the G2/M cell cycle phase: Lessons learned from the yeast MEN and SIN pathways

“…In this context, one should also note that Hippo mutant cells from dissected developing wings or eyes do not display obvious cell cycle phasing defects [26,55], suggesting that Hippo might not have a role in mitosis in every cell type. However, RasV12 immortalised cells derived from warts null fly embryos display polyploidy, which suggests a defective tetraploidy checkpoint [61]. Given all these indications, future research is warranted to address how Hippo/Lats (and possibly also Trc) signalling plays a role in mitosis.…”

Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway

“…We therefore established primary cell cultures from embryos with Ras activation (Dequéant et al 2015), SWH pathway inactivation, Wts-RNAi, or the combined alterations (Simcox et al 2008). These lines could be grown continuously in culture for >100 passages, and, upon transplantation in vivo, Ras V12 and Wts-RNAi; Ras V12 formed tumors in flies (Wts-RNAi alone was not tested) (Simcox et al 2008; data not shown).…”

“…Fly strains rs,hsFLP,y,w 1118 ;Act5C>CD2>GAL4,UAS-RFP/TM3Sb, UAS-NLS-GFP, UAS-Ras V12 (a gift from G. Rubin), UAS-Wts-RNAi (Simcox et al 2008), and UAS-Wts-RNAi;UAS-Ras V12 stocks were used in this study.…”