Figure 1.

Generation and characterization of bArKO and tArKO mice. (A) Schematic demonstrating generation of the floxed murine aromatase gene and its recombination after expression of recombinases. The upper diagram shows the targeting construct used to introduce loxP and FRT sites into the WT aromatase gene; restriction enzyme sites (Sca1 and EcoRV), the location of 5′ and 3′ probes, and the position of the primers (F1, F2, and R) for the PCR analysis are indicated. The middle diagram shows the targeting construct after homologous recombination. The aromatase allele together with the FRT-flanked neo cassette was flanked by LoxP sites (Arom^fl/neo). The lower diagram indicates the floxed aromatase alleles after transient Flp recombinase expression with subsequent deletion of the neo cassette (Arom^fl). Nestin-Cre recombinase expression with knockout of the flanked region of the aromatase gene (Arom^ko) in the brain generated bArKO mice, and Zp3-Cre recombinase expression with complete deletion of the flanked region of the aromatase gene (Arom^del) in whole body generated tArKO mice. I.f, a brain-specific exon 1. PII, a gonad-specific first exon. E2, exon 2. E3-E10, exon 3 to exon 10. (B) Southern blot analysis of different ES cell clones with the 5′ probe and 3′ probe after transfection with the LoxP/FRT flanked targeting construct. Clones #3 and #4 showed the expected bands of 18.9 kb (Arom^wt allele) and 6.9 kb (Arom^fl/neo allele) using the 5′ probe and the expected bands of 14.4 kb (Arom^wt allele) and 8.2 kb (Arom^fl/neo allele) using the 3′ probe, indicating that 1 copy of the WT aromatase gene was replaced by the targeting construct. (C) PCR analysis of DNA prepared from brain and testis of bArKO and tArKO mice. bArKO mice produced a 263-bp band (Arom^ko allele) in brain indicating a recombination event in brain and a 182-bp band (Arom^fl allele) in testis indicating a loxP-flanked aromatase gene without recombination in testis. Aromatase heterozygous control mice contained an Arom^wt allele (a 215-bp band) and an Arom^ko allele in brain, and an Arom^wt allele and an Arom^fl allele in testis. tArKO mice contained only the Arom^del allele (a 263-bp band) in brain and testis suggesting that recombination occurred in brain and testis. Aromatase heterozygous control mice (Het) contained an Arom^wt allele and an Arom^del allele in brain and testis. WT mice only contained the Arom^wt allele. (D) Real-time quantitative PCR demonstrating mRNA levels of aromatase-expressing tissues in control, bArKO, and tArKO mice. GAPDH mRNA levels served as loading controls. n = 5. E₂ levels in the brain (E and G) and testis (F and H) of bArKO and tArKO mice were measured by the LC-MS/MS assay. 2-tailed Student t test or 1-way ANOVA with Tukey multiple comparison test, *P < 0.05, **P < 0.01. n = 9-12 for controls and n = 6 for bArKO mice; n = 21 for WT, n = 25 for Het, and n = 16 for tArKO mouse brain; n = 9 for WT, n = 6 for Het, and n = 6 for tArKO mouse testis.