Generating CRISPR-edited clonal lines of cultured Drosophila S2 cells.

CRISPR/Cas9 genome editing is a pervasive research tool due to its relative ease of use. However, some systems are not amenable to generating edited clones due to genomic complexity and/or difficulty in establishing clonal lines. For example, Drosophila Schneider 2 (S2) cells possess a segmental aneuploid genome and are challenging to single-cell select. Here, we describe a streamlined CRISPR/Cas9 methodology for knock-in and knock-out experiments in S2 cells, whereby an antibiotic resistance gene is inserted in-frame with the coding region of a gene-of-interest. By using selectable markers, we have improved the ease and efficiency for the positive selection of null cells using antibiotic selection in feeder layers followed by cell expansion to generate clonal lines. Using this method, we generated the first acentrosomal S2 cell lines by knocking-out centriole genes Polo-like Kinase 4/Plk4 or Ana2 as proof of concept. These strategies for generating gene-edited clonal lines will add to the collection of CRISPR tools available for cultured Drosophila cells by making CRISPR more practical and therefore improving gene function studies.

Chromatin Immunoprecipitation (ChIP) of Heat Shock Protein 90 (Hsp90).

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique for genome-wide mapping of protein-DNA interactions and epigenetic marks in vivo. Recent studies have suggested an important role of heat shock protein 90 (Hsp90) in chromatin. This molecular chaperone assists other proteins to acquire their mature and functional conformation and helps in the assembly of many complexes. In this chapter, we provide specific details on how to perform Hsp90 ChIP-seq from Drosophila Schneider (S2) cells. Briefly, cells are simultaneously lyzed and reversibly cross-linked to stabilize protein-DNA interactions. Chromatin is prepared from isolated nuclei and sheared by sonication. Hsp90-bound loci are immunoprecipitated and the corresponding DNA fragments are purified and sequenced. The described approach revealed that Hsp90 binds close to the transcriptional start site of around one-third of all Drosophila coding genes and characterized the role of the chaperone at chromatin.

Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components.

BACKGROUND: BMP signaling is involved in myriad metazoan developmental processes, and study of this pathway in Drosophila has contributed greatly to our understanding of its molecular and genetic mechanisms. These studies have benefited not only from Drosophila's advanced genetic tools, but from complimentary in vitro culture systems. However, the commonly-used S2 cell line is not intrinsically sensitive to the major BMP ligand Dpp and must therefore be augmented with exogenous pathway components for most experiments. RESULTS: Herein we identify and characterize the responses of Drosophila ML-DmD17-c3 cells, which are sensitive to Dpp stimulation and exhibit characteristic regulation of BMP target genes including Dad and brk. Dpp signaling in ML-DmD17-c3 cells is primarily mediated by the receptors Put and Tkv, with additional contributions from Wit and Sax. Furthermore, we report complex regulatory feedback on core pathway genes in this system. CONCLUSIONS: Native ML-DmD17-c3 cells exhibit robust transcriptional responses to BMP pathway induction. We propose that ML-DmD17-c3 cells are well-suited for future BMP pathway analyses.

Chromatin Immunoprecipitation (ChIP) of Heat Shock Protein 90 (Hsp90).

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique for genome-wide mapping of protein-DNA interactions and epigenetic marks in vivo. Recent studies have suggested an important role of heat shock protein 90 (Hsp90) at chromatin. This molecular chaperone assists other proteins to acquire their mature and functional conformation and helps in the assembly of many complexes. In this chapter, we provide specific details on how to perform Hsp90 ChIP-seq from Drosophila Schneider (S2) cells. Briefly, the cells are simultaneously lyzed and reversibly cross-linked to stabilize protein-DNA interactions. Chromatin is prepared from isolated nuclei and sheared by sonication. Hsp90-bound loci are immunoprecipitated and the corresponding DNA fragments are purified and sequenced. The described approach revealed that Hsp90 binds close to the transcriptional start site of around one-third of all Drosophila coding genes and characterized the role of the chaperone at chromatin.