Inducible model for beta-six-mediated site-specific recombination in mammalian cells

Abstract

The prokaryotic beta recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in chromatin-integrated substrates. Here, we demonstrate that an enhanced green fluorescent protein (EGFP)-fused version of beta recombinase (beta-EGFP) is fully active, retaining most specific activity. It is used to develop a recombination-dependent activatable gene expression (RAGE) system based on the androgen receptor (AR) ligand-binding domain (LBD). Two hybrid molecules, a direct fusion of the LBD-AR to the C-terminus of beta recombinase (beta-AR) and a triple fusion of beta-EGFP to the same ligand-binding domain (beta-EGFP-AR), were engineered and their subcellular behavior, stability and catalytic activity were evaluated. Both chimeric beta recombinase proteins showed in vivo inducible recombinogenic activity dependent on addition of an androgen receptor agonist, although the beta-AR fusion protein demonstrated more accurate ligand-dependent translocation from cytoplasm to nucleus.

Figure 1

β Recombinase expression using retroviral vectors. (A) Scheme of LZR-β-EGFP and LZR-β-ires-EGFP retroviral vectors used. Expression of a direct fusion protein between β recombinase and EGFP (upper); independent expression of both proteins using an IRES (lower). Arrows indicate the transcribed sequence. (B) NIH-3T3 cells were transduced with both vectors; after 48 h, total proteins were extracted. Western blot autoradiograph shows expression of the fusion protein (β-EGFP, 50 kDa, left lane), individual proteins (β, 23.4 kDa; EGFP, 27 kDa, right) and 5 ng of purified β recombinase (center). Non-specific bands are marked with an asterisk on the membranes. (C) Immunofluorescence of NIH-3T3 cells transduced with LZR-β-EGFP (upper) and LZR-β-ires-EGFP (lower), showing nuclear localization of β recombinase, fused or alone. EGFP is detected in the same nuclear dots when LZR-β-EGFP is used, but not when β recombinase and EGFP are expressed independently (LZR-β-ires-EGFP). TOPRO stains nuclei.

Figure 2

Retroviral vector potential in regulated expression systems. (A) Scheme for target plasmids used. psps-Luc plasmid, in which a puromycin gene is located between two directed six sites under the control of an EF-1α promoter (upper). A luciferase gene is inserted downstream, followed by a hygromycin resistance gene. β Recombinase expression causes deletion of the puromycin gene including its stop codon and one six site, allowing luciferase reporter gene expression. Positive control plasmid (pLuc) with direct luciferase expression, under the same promoter (lower). Arrows indicate transcription units and arrowheads indicate specific primers used to detect recombination. (B) NIH-3T3 cells were electroporated with psps-Luc and pLuc, and hygromycin-resistant cells were selected. psps-Luc NIH-3T3-resistant cells were transduced with β-EGFP or β-ires-EGFP retroviral vectors; luciferase expression was measured after 48 h. The negative sample (neg) involves transduction of psps-Luc NIH-3T3-resistant cells with a control vector.

Figure 3

Analysis of recombination in psps-Luc-transduced NIH-3T3 clones. Ten clones (1–10) were isolated and SSR analyzed by PCR and luciferase expression. (A) Genomic DNA of each clone (−, untransduced cells; +, transduced cells) was amplified using specific primers of the EF-1α promoter and luciferase gene (see Figure 2A). Non-recombined DNA (NR; 1.5 kb band), recombined DNA (R; 0.4 kb band) (upper panel). The recombination level was determined by luciferase activity quantification (lower panel). (B) EGFP-positive subpopulations were purified by cell sorting of three different clones (7, 8 and 10). Recombination level in untransduced (−) and transduced (pre-sorting and post-sorting) cells was analyzed by PCR.

Figure 4

Analysis of recombination time-course. Several C10 subclones were obtained and their recombination rates analyzed over a 7-week period. The recombination rate obtained for five of them (10.2, 10.3, 10.5, 10.6 and 10.7) was analyzed by several techniques. (A) Non-recombined (NR) and recombined (R) DNA fragments amplified by PCR. (B) Detection of β recombinase expression and histone H1, as control, by western blot. (C) Luciferase activity. Western blot and luciferase activity were measured in the fifth week.

Figure 5

SSR using an inducible system based on fusion of β recombinase and the androgen receptor. (A) Scheme showing the retroviral vectors used in the inducible system. LZR-β-EGFP-(AR)LBD vector expresses the triple fusion protein (β-EGFP-LBD) of β recombinase, EGFP and the androgen receptor LBD (upper); the lower panel shows LZR-β-(AR)LBD-ires-EGFP vector which allows independent expression of the β-LBD fusion protein and EGFP by an IRES. Arrows indicate transcription units. (B) C7, C8 and C10 clones were transduced with the retroviral vectors and the EGFP-positive subpopulation selected by cell sorting. Western blot shows expression of β-EGFP-LBD (80 kDa), β-LBD (54 kDa), EGFP (27 kDa), and tubulin (control, 55 kDa) in transduced cells, untreated (−) or treated with (+) mibolerone (Mib; 10⁻⁸ M, 48 h). Non-specific bands are marked with an asterisk on the membranes. (C) Immunocytochemistry of β-LBD-ires-EGFP-expressing cells, alone or mibolerone-treated. (D) Immunocytochemistry of β-EGFP-LBD-transduced cells, alone or with mibolerone. Only data obtained in clone C8 have been included in (B–D). Clones C7 and C10 rendered quite similar results.

Figure 6

Analysis of luciferase expression using the inducible system. C7, C8 and C10 clones were transduced with these retroviral vectors and the EGFP-positive subpopulation selected by cell sorting. Graphs show luciferase expression in function of induction time in culture with 10⁻⁸ M mibolerone. (A) Luciferase expression of β-LBD-ires-EGFP-transduced clones. (B) Luciferase expression of β-EGFP-LBD transduced clones.