Drosophila embryonic 'fibroblasts': Extending mutant analysis in vitro

Simcox, Amanda; Austin, Christina L; Jacobsen, Thomas; Jafar‐Nejad, Hamed

doi:10.4161/fly.7427

Cited by 16 publications

(26 citation statements)

References 5 publications

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“…To study miRNA processing in the complete absence of Loqs, we generated a cell line from loqs null mutant embryos ( loqs-KO , a deletion allele) following a protocol for establishing Drosophila cell lines using expression of oncogenic Ras V12 (Liu et al, 2007; Simcox et al, 2008a; Simcox et al, 2008b). Only cells mutant for loqs also express Ras V12 , which gives them a proliferation advantage (Figure 1A) (Simcox et al, 2008a).…”

Section: Resultsmentioning

confidence: 99%

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site

Lim

Chou

et al. 2016

Cell Reports

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In Drosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major co-factor. Previous analyses indicated that loqs mutants only partially impede miRNA processing but the activity of minor isoforms or maternally deposited Loqs was not eliminated in these studies. We addressed this by generating a cell line from loqs null embryos, and found that only ~40% of miRNAs showed clear Loqs-dependence. Genome-wide comparison of the hairpin structure and Loqs-dependence suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificial alteration of base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in the Loqs-dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs-dependent. We propose that Loqs may have roles in assisting the de novo emergence of miRNA genes by facilitating dicing of suboptimal hairpin substrates.

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Section: Resultsmentioning

confidence: 99%

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site

Lim

Chou

et al. 2016

Cell Reports

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“…For this reason there is no requirement to sort the embryos to select the desired genotype prior to setting up the primary cultures (see Note 3 ). The method can easily be adapted to generating mutant cell lines by using genetics to make strains in which only the homozygous mutant cells are also Act5C-GAL4; UAS-Ras V12 (5). While the method primarily gives rise to lines of cells with a spindle shape, two Ras V12 -expressing lines with epithelial character have been generated; one of these expresses both Ras V12 and wts dsRNA (3).…”

Section: Methodsmentioning

confidence: 99%

“…The Drosophila method has already been used to derive cells of a particular mutant strain (5). But its potential to generate cell type-specific cell lines has not been developed.…”

Section: Introductionmentioning

confidence: 99%

Progress Towards Drosophila Epithelial Cell Culture

Simcox

2012

Methods in Molecular Biology

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Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens.

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“…We have successfully used expression of transgenic Ras V12 to derive cell lines from the rumi mutant [8]. The cells, which are null for rumi have been used to study Notch signaling in vitro [9].…”

Section: Resultsmentioning

confidence: 99%

Loss of the Tumor Suppressor Pten Promotes Proliferation of Drosophila melanogaster Cells In Vitro and Gives Rise to Continuous Cell Lines

et al. 2012

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In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (RasV12) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing RasV12. Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines.

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Drosophila embryonic 'fibroblasts': Extending mutant analysis in vitro

Cited by 16 publications

References 5 publications

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site

Progress Towards Drosophila Epithelial Cell Culture

Loss of the Tumor Suppressor Pten Promotes Proliferation of Drosophila melanogaster Cells In Vitro and Gives Rise to Continuous Cell Lines

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