Tools for Targeted Genome Engineering of EstablishedDrosophilaCell Lines

Abstract

We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies.

Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality inDrosophila cells

“…As an alternative strategy, we assessed the efficiency of phiC31 site-specific recombination mediated by plasmid transfection. Methods for recombination in cell lines were recently developed (20)(21)(22), but the quantitative efficiency of integration has not been reported. Efficiency is critical for pooled screening applications as it must reach a threshold above which generating and maintaining >1000X representation of a library of tens of thousands of elements becomes technically and costfeasible (23).…”

Generating and working with Drosophila cell cultures: Current challenges and opportunities

“…As a result, the copy number of the integrated DNA, and therefore the strength of transgene expression cannot be easily controlled, even after single cell cloning. To address this issue, there were significant efforts to create new and engineer existing cell lines such that it has become possible to stably insert single copy transgenes at known genomic loci in cell lines (Cherbas et al, ; Manivannan et al, ). This is important especially for experiments that assay for the effects of different transgenes, so that transgenes can be expressed at comparable levels.…”

“…Using P‐element transformation, Cherbas et al () transformed existing Kc167 and Sg4 lines to carry a single attP‐flanked GFP cassette at multiple random loci. In contrast, Manivannan et al () generated de novo Ras‐attP1 and Ras‐attP2, two act‐GAL4 driven Ras V12 cell lines derived from flies that harbor existing attP‐flanked cassettes at known genomic loci.…”

“…Both approaches demonstrated successful RMCE facilitated by ϕC31 integrase (Bateman, Lee, & Wu, ) via the use of targeting constructs containing attB sites. The resulting stable cell lines carry single copy transgenes of inducible mCherry , Tubulin, Jupiter or Cas9 at defined genomic loci (Cherbas et al, ).…”

“…Drug sensitivity can enrich for the desired cells after transfection in order to establish stably transformed lines. Depending on the selection marker, drugs such as puromycin, blasticidin, methotrexate, or hygromycin have been used to select desired cells, while thymidine kinase‐conferred sensitivity to Ganciclovir has been used to remove cells with illegitimate insertions (Cherbas et al, ; Franz, Brunner, et al, ; Franz, Shlyueva, et al, ; Kunzelmann et al, ; Manivannan et al, ). Alternatively, cells carrying fluorescence markers can be selected by flow cytometry (Neumuller et al, ).…”