Table 3. Quantitative RT–PCR analyses of E12 +/2 versus +/+ B6 XYAKR gonads and E14 +/2 B6 XYAKR ovaries (ovotestes excluded) versus +/+ B6 XYAKR testes. Table 3. Cont. Fold change in Wt1+/2 Significant (p,0.05) Stage Gene Fold change in Sf1+/2 Significant (p,0.05) Modeling Dosage-Sensitive Sex Reversal in Mice G G, T G, T G T T G, T G, T T T T T T P = 0.06 G, T G, T G, T G, T G, T T G, T G, T G, T G, T G, T Gene Bmp2 Cbln4 Dhh Mro Ptgds Defb19 Adamts19 Bmp2 Fgfr2 Fst Sry Wnt4 Aard Amh Cbln1 Cbln4 Col2a1 Col9a3 Cst9 Cyp11a1 Cyp17a1 Cyp26b1 Dhh Etd Hhip Mro Ptgds Ren1 Sostdc1 Sox9 Defb19 Gene Bmp2 Sostdc1 Aard Amh Cbln4 Cst9 Dax1 Dhh Hsdb1 Ptgds Sf1 StAR Sox9 Defb19 Adamts19 Bmp2 2.4 25.4 26.0 26.0 211.82 23.4 10.7 10.7 2.4 15.5 9.6 12.1 24.1 220.8 23.3 23.9 22.1 26.9 25.1 24.7 28.2 24.0 27.1 218.3 24.1 22.1 28.0 24.8 24.7 22.0 28.6 2.3 2.4 29.9 219.9 27.3 222.6 22 213.1 21.9 212.7 22.1 21.9 22.3 28.7 6.8 7.2 T G, T G, T G, T G, T T G, T G, T G G, T G, T G, T G, T G, T G, T G, T G G, T G, T G, T G, T G, T G, T T G, T G, T G, T G, T G, T G, T G, T T G, T G, T G, T G G, T T G, T T G, T G T G T G, T G, T Fold change in Sf1+/2 Significant (p,0.05) Stage E12 E14 Stage E12 E14 PLoS Genetics | www.plosgenetics.org 8 April 2012 | Volume 8 | Issue 4 | e1002569 Fgfr2 Fst Sry Wnt4 Aard Amh Cbln1 Cbln4 Col9a3 Cst9 Cyp11a1 Cyp17a1 Cyp26b1 Dhh Etd Hhip Hsdb1 Mro Ptgds Ren1 Scarb1 Sostdc1 Sox9 Defb19 Vnn1 2.9 25.9 56.9 3.0 23.4 229.5 29.6 24.3 24.6 27.3 266.9 289.9 28.3 29.1 222.3 26.1 214.7 22.4 215.4 228.9 22.8 28.2 23.2 218.5 22.6 G = GPR score equal or greater than 0.4. T = Student’s t-test. doi:10.1371/journal.pgen.1002569.t003 The Wt1 and Sf1 B6 alleles require additional interacting genes to be homozygous for the B6 allele to function as hypomorphs The results presented above suggest that the Wt1B6 and Sf1B6 alleles are hypomorphic alleles. One possibility is that the Wt1B6 and Sf1B6 alleles behave as intrinsic hypomorphs. An alternative possibility is that the Wt1B6 and Sf1B6 alleles function as hypomorphs only when one or more additional interacting gene(s) are homozygous for B6 alleles. To distinguish between these two alternatives, genetic crosses were designed that would generate hybrid (D26B6)F1 XYAKR mice that are heterozygous for the (knockout) allele of Wt1 or Sf1 and the Wt1B6 or Sf1B6 allele, respectively (e.g. Wt1KO/Wt1B6 or Sf1KO/ Sf1B6 ) (Table 2). These F1 hybrid mice are genetically identical to Wt1+/2 and Sf1+/2 B6 XYAKR mice at the Wt1 and Sf1 loci; however, the remainder of the genome is different because it is (D26B6)F1 hybrid (except for the regions immediately around the Sf1KO and Wt1KO alleles that are derived from the 129 strain). Because Wt1+/2 and Sf1+/2 (D26B6)F1 XYAKR mice are not sex reversed, we conclude that the Wt1B6 and Sf1B6 alleles function as hypomorphs only when other genes (modifier genes) are homozygous for B6 alleles. Molecular analysis of the difference between the B6 and D2 alleles of the Wt1 and Sf1 genes The B6 and D2 alleles of the Wt1 and Sf1 genes could encode different protein variants and/or have different expression