TY - JOUR T1 - Targeted insertion in well-characterized <em>Drosophila</em> cell lines using φC31 integrase JF - bioRxiv DO - 10.1101/024216 SP - 024216 AU - Lucy Cherbas AU - Jennifer Hackney AU - Lei Gong AU - Claire Salzer AU - Eric Mauser AU - Dayu Zhang AU - Peter Cherbas Y1 - 2015/01/01 UR - http://biorxiv.org/content/early/2015/08/08/024216.abstract N2 - 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 and 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 demonstrated the technology by integrating a cassette containing a Cu++-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. ER -