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Figure 2
Generation and characterization of conditional calcrl line. (A) Schematic diagram depicting strategy used for generation of a floxed calcrl allele by gene targeting. The top figure shows the endogenous wild-type calcrl allele. The targeting vector was designed so that loxP sites would flank the same exons that were deleted in the calcrl global knockout (36). The third line depicts the targeted calcrl^Flox allele, and the fourth line depicts the calcrl^LoxP allele after Cre-mediated excision. Primers used for isolation of correctly targeted ES cells and for routine genotyping are indicated by small arrows. (B) Correctly targeted ES cells were confirmed by Southern blot analysis using the probe depicted in A. (C) PCR genotyping for the calcrl^Flox allele. (D) Quantitative RT-PCR was performed on RNA isolated from lungs and hearts of wild-type and homozygous calcrl^Flox/Flox mice and revealed no significant differences in the expression of the calcrl^Flox allele before Cre-mediated excision. (E) Schematic representation of breeding scheme used to generate mice with calcrl expression deleted specifically in endothelial cells by use of the Tie2Cre transgene. (F) Results of cross demonstrate that no viable calcrl^LoxP/–Tie2Cre⁺ mice were found beyond E16.5. (G) Compared with calcrl^LoxP/+Tie2Cre⁺ control littermates, the calcrl^LoxP/–Tie2Cre⁺ mice displayed remarkable hydrops without hemorrhage, which phenocopied the global calcrl–knockout phenotype, yet often occurred substantially later, at E16.5. Original magnification, ×10.