Regulation of Cre recombinase by ligand-induced complementation of inactive fragments

Abstract

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.

Figure 1

Principles of the DiCre system. (A) Scheme of the DiCre system. The enzyme is split into two fragments without enzymatic activity, that are fused to proteins (here FKBP12 and FRB) that can be dimerized by a small molecule ligand. Dimerization leads to the association of the complementing Cre moieties and the reconstitution of enzymatic activity. (B) Ribbon/cylinder model of Cre recombinase, based on the published structure (23). Arrows point to the sites of splitting within the interhelical loops B/C (1) or D/E (2). The Tyr324 residue in the active site within the C-terminal domain has been colored. (C) Schematic representation of the constructs used for DiCre. The translation initiation codon has been placed into the context of Kozak’s consensus sequence (36). NLS corresponds to a nuclear localization signal peptide (37). The unique NheI (N) and BamHI (B) sites flanking the linker sequence allow its simple replacement with variants. (D) List of the different linkers tested. Sequences were defined to obtain flexible (38) (linkers F1–F6) or α-helical [AAAKE motif (38); linkers H1–H3] peptides of variable length. The lengths correspond to that of the maximally extended configuration of the linker peptide. (E) Conceptual model of DiCre within the Holliday structure. The insert shows the scheme of the planar Holliday junction, with the four Cre monomers bound to the half LoxP sites. The different portions of the fusion molecules are assembled in distinct planes that are parallel to that of the DNA.

Figure 2

Analysis of DiCre by transient transfection. (A) Scheme of the vector used to establish the Rat2/CALNLZ indicator cell line. The transcription of the CALNLZ unit (28) is driven by the CAG promoter. LacZ is transcribed only if a floxed fragment comprising the aminoglycosyd phosphotransferase (neo) gene with a translational stop codon and polyadenylation signal is excised. ‘Probe’ corresponds to the 890 bp fragment used for Southern blots. Neo allows the selection of infected cells. The restriction sites indicated correspond to those used to examine viral integration (B) or recombination (EcoRI–ApaI; Fig. 3D). (B) Southern blot analysis of viral integration of the Rat2/CALNLZ clone 1 used throughout this study. Genomic DNA was digested with EcoRI, HindIII or HpaI. The existence of a single band in each case indicates a single insertion. (C) Example of the recombination in Rat2/CALNLZ cells following transient transfection with different Cre constructs. The example shown corresponds to the results obtained with pairs that were used thereafter to characterize the DiCre system, i.e. 59.F2–60.F2 and 104.F5–106.F5. CrePR1 corresponds to a previously described Cre construct regulated by RU-486 (12). Cre activity has been induced by 50 nM rapamycin (DiCre constructs) or 1 µM RU-486 (CrePR1). (D) Comparison of the level of rapamycin-induced recombination following the transient transfection of the different pairs of complementing Cre fragments. The number of X-gal-positive cells/well was expressed as the percentage of the maximal value obtained (with the 104.F4–106.H2 combination). The percentages were coded by levels of gray as indicated on the right. The results correspond to the mean of four experiments.

Figure 3

Analysis of DiCre expressing stable Rat2/CALNLZ transformants. (A) Southern blot analysis of the integration for the Cre constructs in clones displaying different levels of background activity. The background values (percentage of X-gal⁺ cells in the absence of dimerizer) for the clones are 59(F2)/60(F5) clones: 13/2F9, 0.1%; 14/1D2, 0.1%; 14/2G6, 0.4%; 14/1G8, 0.05%; 104.F6–106.F6 clone 2/2C3, 25.1; 104.F5–106.F5 clones, 4/2E3, 3.1%; 4/1A2, 36%; 59/60 clone 12/C8, 0.7 %; 104/106 clone 18/D12, 100%. To analyze the 59(F2), 104(F5), 104(F6), Cre(59) and Cre(104) constructs, hybridization was performed with a probe homologous to a 760 bp sequence of the puromycin (Puro) resistance gene of the pQCXIP bicistronic vector, while for the 60(F2), 106(F5), 106(F6), Cre(60) and Cre(106) constructs, an RNA probe of 1030 nucleotides, homologous to the hygromycin (Hygro) resistance gene of the pQCXIH bicistronic vector, was used. For each clone, a single band, indicative of a single insertion, was obtained for both constructs. (B) Northern blot analysis of the level of transcription of the transduced Cre construct in the clones analyzed in (A). The same probes were used as in (A) with an additional 630 bp probe homologous to cyclophilin A (Cyc). The differences in mRNA levels found between the different clones are relatively weak and do not correlate with the level of background activity. (C) Dose–response curves for rapamycin and AP23102. Cells were exposed to the indicated concentrations of the dimerizer for 72 h and the percentage of recombined (X-gal-positive) cells was evaluated by direct counting. +FK corresponds to the level of excision observed when FK-506 is added with the dimerizer (200 nM against rapamycin and 500 nM against AP23109). The results shown correspond to the means of three experiments performed each time on three different 59.F2–60.F2 clones. (D) Kinetics of recombination in 59.F2–60.F2 cells (clone 14/1G8) following exposure to 10 nM AP23102. The dimerizer was added at t = 0 and removed at the indicated times. Genomic DNA was prepared either immediately after the removal of the dimerizer or at t = 72 h (lanes 3+, 6+, 12+, 18+ and 24+). Hybridization was performed with a probe homologous to an internal segment of the provirus (Fig. 2A.); NE corresponds to the fragment obtained when no recombination has occurred; E is the fragment obtained after recombination. (a) Kinetics of recombination when the dimerizer is present continuously for the indicated length of time. (b) Level of recombination observed at t = 72 h with the dimerizer present only for the indicated length of time, beginning at t = 0. (E) Cell proliferation following the transient activation of DiCre by AP23102. Cells were exposed to 10 nM of the dimerizer for 24 h. The cells were cultured for a further 2 (group +2d) or 6 (group +6d) days before the evaluation of proliferation over a subsequent 3-day period. Controls correspond to the cells not exposed to the dimerizer; AP23102 correspond to cells exposed to 10 nM AP23102 only during the period of evaluation of cell proliferation. The results shown are the means obtained with four different 59.F2–60.F2 clones.