Cre reconstitution allows for DNA recombination selectively in dual-marker-expressing cells in transgenic mice

Abstract

Cre/LoxP-based DNA recombination has been used to introduce desired DNA rearrangements in various organisms, having for example, greatly assisted genetic analyses in mice. For most applications, single gene promoters are used to drive Cre production for conditional gene activation/inactivation or lineage-tracing experiments. Such a manipulation introduces Cre in all cells in which the utilized promoter is active. To overcome the limited selectivity of single promoters for cell-type-specific recombination, we have explored the 'dual promoter combinatorial control' of Cre activity, so that Cre activity could be restricted to cells that express dual protein markers. We efficiently reconstituted Cre activity from two modified, inactive Cre fragments. Cre re-association was greatly enhanced by fusing the Cre fragments separately to peptides that can form a tight antiparallel leucine zipper. The co-expressed Cre fusion fragments showed substantial activity in cultured cells. As proof of principle of the utility of this technique in vivo for manipulating genes specifically in dual-marker-positive cells, we expressed each inactive Cre fragments in transgenic mice via individual promoters. Result showed the effective reconstitution of Cre activates LoxP recombination in the co-expressing cells.

Figure 1.

A diagram of the half-Cre molecules and the interacting peptide sequences. (A) The Cre molecule was designed to be cleaved into two molecules between two glycine residues (amino acid residues 190–191, as numbered in X03453). The N-terminal half was fused with one of a pair peptides that form a leucine zipper (N-peptide), whereas the C-terminal half was fused with the other peptide (C-peptide). (B) The C- and N-peptide sequences.

Figure 2.

Assisted Cre reconstitution restores substantial Cre activity in cell culture. (A) The structure of the Cre reporter. EGFP could be expressed only after Cre-mediated excision of the stop signal. (B1–B3) One example of Cre activity assays. (B1) EGFP expression (green) reported detectable Cre activity. (B3) mCherry expression (magenta) indicates transfected cells. (B2) Merge of B1 and B3. (C) An example of flow cytometry analysis for Cre activity (0.5 ng pCIG-Cre plasmid/well). In the four quadrants (R1–R4), R1 represents untransfected cells; R2, transfected cells without detectable Cre activity; R3, transfected cells with active Cre; R4, cells with active Cre yet have lost mCherry expression. The percentage of each cell type is labeled. The ratio of R3/(R2+R3) is used as Cre activity index. (D) The plot of GFP⁺/mCherry⁺ cells [=R3/(R2+R3)] versus the amount of Cre-expressing plasmid. (E) Four assays for the relative Cre activity reconstituted from various Cre fragments. The four DNA samples used were (labeled below each dot plot): no Cre control, pCIG-αCre + pCIG-βCre-nls, pCIG-nCre + pCIG-nlcCre; pCIG-nCre + Ubc-nlcCre (all plasmids were transfected at 2 ng/well). Note the low GFP background when no Cre activity is present. (F) Relative Cre activity restoration when different Cre fragments are utilized for complementation. (G) Relative Cre activity with a combination of 1 ng of pCIG-nCre with various amount of pCIG-nlcCre.

Figure 3.

Transgene structures and a mouse cross scheme. (A) DNA constructs that utilize a Pdx1 promoter to drive the expression of nCre and nlcCre. (B) An example of mouse cross scheme to produce animals of desired genotype.

Figure 4.

Reconstituted Cre activates reporter gene expression in transgenic mice. Green fluorescence is from YFP, indicating cells that have undergone Cre-mediated recombination. (A) E13.0 nCre^tg1; R26YFP pancreas. The green channel in this picture was enhanced to visualize the lobular pancreatic structure in the dorsal pancreas. (B and C) The dorsal and ventral lobe of a nCre^tg1; nlcCre^tg2; R26YFP pancreas (E13.0). (D and E) The dorsal and ventral lobe of a nCre^tg1; nlcCre^tg2; R26YFP pancreas (E17.5). (F) The ventral pancreas of a E17.5 nCre^tg1; nlcCre^tg2; R26YFP/R26YFP animal. (G–I) Pancreatic regions of Pdx1-Cre^tg; R26YFP animals. (G) E10.5; (H) E13.0; (I) Neonatal. Note the presence of YFP⁺ cells in the duodenum (F). dp, dorsal pancreas; vp, ventral pancreas; du, duodenum. Bars = 40 μm.

Figure 5.

Cre activity can be reconstituted in all pancreatic progenitors. Green fluorescence indicated YFP⁺ cells. Shown were pancreatic regions only. Magenta fluorescence indicated the expression of pancreatic markers, as marked in each panel. (A–F) Neonatal pancreas. (G–I) Two-month-old adult pancreas. Yellow arrows, double positive cells. amy, amylase; ins, insulin; SS, somatostatin; DBA, Dolichos-Biflorus agglutinin; glc, glucagon; PP, pancreatic polypeptide. Bar = 20 μm.