Fig 1.
Construction of Cyp2b9/10/13-null mice.
(A) Cyp2b9/10/13-null mice were produced using Crispr/Cas9 with sgRNA target sites for all three genes. A 287 kb deletion mutant was produced that lacks these three Cyp2b genes found in tandem repeat on chromosome 7. (B) Two mice heterozygote for a chromosome deletion lacking the three Cyp2b genes (Cyp2b10, 2b13, 2b9) in tandem repeat were produced. PCR confirmation of the 287kb deletion from the first null mice produced is shown in lanes 4 and 21 using the F2/R2 primer combination that produces a 1066 bp fragment. (C) Subsequent breeding produced mice lacking Cyp2b9/10/13 as demonstrated by the presence of the 1066 bp PCR product. (D) Heterozygotes were discerned from homozygotes by PCR of Cyp2b13 indicating a heterozygote.
Table 1.
Compensatory changes in CYP gene expression in CAR-null mice.
Table 2.
Compensatory changes in CYP gene expression in Cyp3a-null mice.
Table 3.
Compensatory changes in CYP gene expression in Cyp2b9/10/13-null mice.
Fig 2.
Compensatory changes in CYP protein expression in CAR-null mice.
Western blots of male and female CAR-null mice show significant changes in CYP expression relative to their WT counterparts. Results are expressed as relative mean of the WT compared to CAR-null mice of the same sex. Statistical differences were determined by Student’s t-tests (n = 2) with * (p < 0.05) ** (p < 0.01) indicating significant differences.
Fig 3.
Testosterone hydroxylation is perturbed in CAR-null mice in a gender-specific manner.
(A) Testosterone hydroxylation was determined in male and female WT and CAR-null mice as described in the Materials and Methods. Data are presented as mean specific activity (μmol/min/mg protein) ± SEM (n = 5). (B) Ratio of 6α/15α-hydroxytestosterone as a biomarker of CYP sexual dimorphism in the liver. An aindicates a significant difference between WT male and Cyp2b9/10/13-null male mice, bindicates a significant difference between WT female and Cyp2b9/10/13-null female mice, cindicates a significant difference between male and female WT mice and dindicates a significant difference between the male and female Cyp2b9/10/13-null mice. Statistical differences were determined by two-way ANOVA followed by Fisher’s LSD as the post-hoc test in (A) and one-way ANOVA followed by Fisher’s LSD in (B). A letter without an asterisk indicates a significance of p < 0.05, asterisk indicate significance of *p<0.01, **p<0.001, and *** p<0.0001, respectively.
Fig 4.
Compensatory changes in CYP protein expression in Cyp3a-null mice.
Western blots of male and female Cyp3a-null mice show significant changes in CYP expression relative to their WT counterparts. Results are expressed as relative mean of the WT compared to CAR-null mice of the same sex. Statistical differences were determined by Student’s t-tests (n = 3) with * (p < 0.05) ** (p < 0.01) *** (p < 0.001) indicating significant differences.
Fig 5.
Changes in testosterone hydroxylation in Cy3a-null mice.
(A) Testosterone hydroxylation was determined in male and female WT and Cyp3a-null mice as described in the Materials and Methods. Data are presented as mean specific activity (μmol/min/mg protein) ± SEM (n = 4). (B) Ratio of 6α/15α-hydroxytestosterone as a biomarker of CYP sexual dimorphism in the liver. An aindicates a significant difference between WT male and Cyp2b9/10/13-null male mice, bindicates a significant difference between WT female and Cyp2b9/10/13-null female mice, cindicates a significant difference between male and female WT mice and dindicates a significant difference between the male and female Cyp2b9/10/13-null mice. Statistical differences were determined by two-way ANOVA followed by Fisher’s LSD as the post-hoc test in (A) and one-way ANOVA followed by Fisher’s LSD in (B). A letter without an asterisk indicates a significance of p < 0.05, asterisk indicate significance of *p<0.01, **p<0.001, and *** p<0.0001, respectively.
Fig 6.
CYP protein expression in WT and Cyp2b9/10/13-null mice.
Western blots of male and female Cyp2b9/10/13-null mice show significant changes in CYP expression relative to their WT counterparts. Cyp2a isoforms show two bands as Cyp2a22 is 50kDa in B6 mice and the other Cyp2a isoforms are 56 kDa. Results are expressed as relative mean of the WT compared to CAR-null mice of the same sex. Statistical differences were determined by Student’s t-tests (n = 3) with * (p < 0.05) *** (p < 0.001) indicating significant differences.
Table 4.
Genes differentially expressed in Cyp2b9/10/13-null female mice compared to WT female mice following microarray analysis.
Fig 7.
Testosterone hydroxylation determined in WT and Cyp2b9/10/13-null mice.
(A) Testosterone hydroxylation was determined in male and female WT and Cyp2b9/10/13-null mice as described in the Materials and Methods. Data are presented as mean specific activity (μmol/min/mg protein) ± SEM (n = 4). (B) Ratio of 6α/15α-hydroxytestosterone as a biomarker of CYP sexual dimorphism in the liver. An aindicates a significant difference between WT male and Cyp2b9/10/13-null male mice, bindicates a significant difference between WT female and Cyp2b9/10/13-null female mice, cindicates a significant difference between male and female WT mice and dindicates a significant difference between the male and female Cyp2b9/10/13-null mice. Statistical differences were determined by two-way ANOVA followed by Fisher’s LSD as the post-hoc test in (A) and one-way ANOVA followed by Fisher’s LSD in (B). A letter without an asterisk indicates a significance of p < 0.05, asterisk indicate significance of *p<0.01, **p<0.001, and *** p<0.0001, respectively.