Targeted insertion in well-characterized Drosophila cell lines using φC31 integrase
mag(2015)
摘要
We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in 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.
更多查看译文
关键词
Drosophila,cell lines,phiC31 integrase,targeted insertion
AI 理解论文
溯源树
样例
生成溯源树,研究论文发展脉络