Generation of genome-modified <i>Drosophila</i> cell lines using SwAP

<p>The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal <i>Drosophila</i> cell lines has remained a longstanding challenge, hampered by the difficulty of getting <i>Drosophila</i> cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered <i>Drosophila</i> cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited <i>armadillo</i> mutant <i>Drosophila</i> cell lines. Our method provides a powerful and simple workflow that improves the utility of <i>Drosophila</i> cells for genetic studies with CRISPR/Cas9.</p

Probing the canonicity of the Wnt/Wingless signaling pathway

<div><p>The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirement for Armadillo (Arm, <i>Drosophila</i> beta-catenin) and Pangolin (Pan, <i>Drosophila</i> TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of <i>Drosophila</i> Kc cells. Using somatic genetics, we demonstrate that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing we identified Wnt/Wg-responsive enhancer elements and found that all responsive enhancers depend on Pan. Together, our results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system.</p></div

Multiorder Correction Algorithms to Remove Image Distortions from Mass Spectrometry Imaging Data Sets

Time-of-flight secondary ion mass spectrometry imaging is a rapidly evolving technology. Its main application is the study of the distribution of small molecules on biological tissues. The sequential image acquisition process remains susceptible to measurement distortions that can render imaging data less analytically useful. Most of these artifacts show a repetitive nature from tile to tile. Here we statistically describe these distortions and derive two different algorithms to correct them. Both a generalized linear model approach and the linear discriminant analysis approach are able to increase image quality for negative and positive ion mode data sets. Additionally, performing simulation studies with repetitive and nonrepetitive tiling error we show that both algorithms are only removing repetitive distortions. It is further shown that the spectral component of the data set is not altered by the use of these correction methods. Both algorithms presented in this work greatly increase the image quality and improve the analytical usefulness of distorted images dramatically

Characterization of the larval hemolymph proteome.

<p>(A) Workflow of the analyses. Hemolymph samples from fed and starved larvae were digested in solution. Tryptic peptides were separated by isoelectric focusing for complexity reduction. Peptides were analyzed using microcapillary liquid chromatography–electrospray ionization–tandem MS (µLC-ESI-MS/MS). SEQUEST spectral search was performed for peptide spectrum matching. (B) Venn diagram illustrating the number of gene models detected in hemolymph from fed and starved larvae, respectively.</p