Tools for Targeted Genome Engineering of Established Drosophila Cell 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 Cu(2+)-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.

Keywords: Drosophila; cell lines; phiC31 integrase; targeted insertion.

Figure 1

Constructs used in this paper. Sequences for these plasmids and for the integrase-expression plasmid are deposited in GenBank. eGFP, DHFR, and HS-TK are each expressed from an Act5C promoter; mCherry is expressed from a MtnA promoter. (A) The P transposons used as docking platforms. Each contains a GFP-expression cassette between parallel attP sites; the encoded fluor is eGFP with a nuclear localization signal. IPPI also contains insulator elements flanking the attP sites. Only the P-element transposons are shown. (B) Vectors for integration by replacement in the docking sites. Each contains a MTX-resistance marker (DHFR) between parallel attB sites for positive selection and a HSV TK-expression cassette outside the attB-bounded region for counter selection against cells that have acquired the entire plasmid by illegitimate integration. The DHFR encoded in these plasmids is resistant to MTX and is of prokaryotic origin (Bourouis and Jarry 1983). HSV TK is a thymidine kinase derived from herpes simplex virus, and its expression renders cells sensitive to GCV. The two vectors differ by the presence in B-DHFR-GW-B-TK of a cassette for inserting constructs using the Gateway system (Life Technologies); B-DHFR-B-TK has a limited number of unique sites for inserting constructs, including an EcRI site upstream of the DHFR transcription unit and a ClaI site downstream of DHFR. (C) A Cu²⁺-inducible mCherry targeting sequence used for integration into the docking sites in experiments described in this paper. The targeting plasmid was made by cloning an Mt-mCherry transcription unit into the Gateway entry vector pENTRB (Life Technologies) and then using the Gateway reaction to place the transcription unit into B-DHFR-GW-B-TK. Only the fragment bounded by attB sites is illustrated here.

Figure 2

Expression of GFP in docking site lines. FACS-generated histograms are shown on the left; fluorescence photomicrographs on the right. Kc167 is the untransformed parental line, exhibiting only autofluorescence. Representative clonal docking site lines are shown below. The vertical blue line at 100 units GFP is provided for visual alignment. GFP-null cells are estimated at about 10% of the population in Kc167-PP-21D and 1% in Kc167-PP-93E. Any GFP-null cells in Kc167-IPPI-66D are masked by the overlapping range of GFP expression. Bar, 25 μm.

Figure 3

Design of a targeted substitution experiment. (A) The desired reaction, catalyzed by phiC31 integrase, in which the act5C-eGFP marker of the docking site is replaced by Mt-mCherry plus a MTX-resistance marker. (B) The result of an illegitimate recombination (not catalyzed by phiC31-integrase) in which the entire targeting plasmid is incorporated at a random chromosomal site. See Table 2 for a summary of the predicted properties of these two transformation products.

Figure 4

Expression of a transgene in transformed clones. mCherry expression (arbitrary units) was measured by FACS analysis and is shown as histograms of cell populations with or without Cu²⁺ treatment (1 mM CuSO₄, 20 hr) and as mean expression and Cu²⁺ induction ratio. (A) Kc167 (parental line); these cells do not contain an mCherry coding sequence; background autofluorescence measured in these cells is subtracted to give the mean mCherry expression estimates shown in the −Cu and +Cu columns. (B and C) Targeted substitution of Mt-mCherry into the docking site lines Kc167-IPPI-66D and Kc167-PP-93E, respectively. (D–F) Illegitimate insertions of the Mt-mCherry plasmid, derived from transformation of Kc167-IPPI-66D (D) or Kc167-PP-93E (E and F) in the absence of φC31-integrase, followed by MTX selection and cloning.