Figure 1.
A: Principle of split-Cre complementation.
NCre- and CCre-coding sequences are placed under the control of two different promoters (promoter 1 and 2, respectively). Only if both promoters are active, functional complementation takes place and LoxP (red triangles) – flanked sequences are recombined, thereby activating reporter genes (A1) or excising exons for knock-out strategies (A2). Ubi: ubiquitously expressed promoter (in our study the ROSA26 promoter). B: Design of fusion proteins. NCre- and CCre-proteins were constructed by fusing amino acids 19–59 (NCre) or amino acids 60–343 (CCre) of the sequence of Cre recombinase to the constitutive active coiled-coil interaction domain of the yeast transcription factor GCN4 (GCN4cc). Immunotags (Flag, Myc), a linker sequence as well as a nuclear localization sequence (NLS) were also added. C–H: Functional complementation of Cre activity in transgenic mice. C–E: NCre and CCre were transgenically expressed in mice using the PLP promoter. Transgenic mouse lines expressing NCre (line PCND) and CCre (line PCCR) were crossed to ROSY reporter mice and analysed for recombination with in the corpus callosum (C). Slices were counterstained with the oligodendroglial marker CC1 (D). The overlay is shown in E. Arrows point to examples of CC1-positive oligodendrocytes, which show also expression of the EYFP reporter. F–H: NCre and CCre were transgenically expressed in mice using the GFAP-promoter. Transgenic mouse lines expressing NCre (line GCNV) and CCre (line GCCF) were crossed to ROSY reporter mice and analysed for recombination in the cerebellum (F). Bergmann glia cells were stained with antibodies against S100β (G). The overlay is shown in H. Arrows highlight S100β-positive cells which show recombination. The scale bar in H corresponds to 50 µm and applies to panels C–H.
Figure 2.
A combination of PLP- and GFAP-promoter drives recombination mainly in astrocytes and NG2-cells.
Transgenic mouse lines expressing NCre under the GFAP promoter and CCre under the control of the PLP promoter were crossed to ROSY-reporter mice and analysed for recombination in different brain regions (A, D, G, J, M). Slices were counterstained using the astroglial marker S100β (B, E, K), the Müller cell marker cellular retinaldehyd binding protein (CRALBP; H) as well as NG2 (N). Overlays are shown in C, F, I, L and O. Most cells showing recombination in these mice stain also for the astroglial marker (examples are marked by arrows in A–L). A subpopulation of EYFP-labeled cells is positive for NG2 (arrow in M–O), but not all NG2 cells express the reporter EYFP (arrow head in M–O). P–S: Developmental appearance of labeled cells in the juxtaventricular zone close to the dorsal 3rd ventricle (P, Q) and in the cerebellum (R, S). Shown are examples from embryonic day 18.5 (P, R) and postnatal day 2 (Q, S). The dotted lines in P, Q show the approximate border of the ventricle. Mouse lines GCNV x PCCK (G–I); GCCF x PCND (M–O), GCNT x PCCK (P–S) and GCNT x PCCR (all others) were used for this analysis. The scale bars in O and S correspond to 50 µm and apply to panels A–O and P–S, respectively. d3V: dorsal third ventricle; GrDG: granular layer of the dentate gyrus; Th: thalamus; cb: cerebellum; ctx: cortex.
Figure 3.
Functional complementation of split-Cre in vivo after viral infection.
Adeno-associated viral vectors (AAV) containing NCre or CCre under the control of the CCK- and GAD67-promoter, respectively, were injected into brains of ROSY reporter mice and recombination was analysed by EYFP expression. A population of interneurons showing DNA recombination was identified (A, D) that express either parvalbumin (B, arrow) or calretinin (E, arrow). Arrowheads mark cells which are positive for the respective marker protein, but show no recombination. The scale bar in F corresponds to 50 µm and applies to all panels. DG: dentate gyrus; hc: hippocampus.