Fbxo24 is predominantly expressed in testes and essential for male fertility.
(A) RT-PCR of Fbxo24 in mouse adult tissues. Fbxo24 is predominantly expressed in the testis. Br: brain, Th: thymus, Lu: lung, He: heart, Li: liver, Sp: spleen, Ki: kidney, Te: testis, Ut: uterus, Ov: ovary, and N.C.: negative control (water). Actb was used as a control. (B) RT-PCR of Fbxo24 using RNAs obtained from mouse testes at various postnatal days. Actb was used as a control. Water was used as a negative control (N.C.). (C) Construction of expression vectors for FBXO24 with (WT) or without (ΔF) the F-box domain. (D) Fbxo24 (WT)-FLAG or Fbxo24 (ΔF)-FLAG was transiently expressed with Skp1-1D4 in HEK293T cells. Immunoprecipitation (IP) was performed using anti-1D4 antibody or anti-FLAG antibody. FBXO24-FLAG interacts with SKP1-1D4 via the F-box domain. α-tubulin was used as a loading control. (E) Schematic for generating Fbxo24 KO mice using the CRISPR/Cas9 system. White boxes indicate untranslated regions while black boxes indicate protein coding regions. The gRNAs used are shown. Fw and Rv indicate the forward and reverse primer used for genotyping, respectively. (F) Genotyping of obtained Fbxo24 mutant mice. Fw #1-Rv #1 primers for KO allele and Fw #2-Rv #2 primers for WT allele in Fig. 1E were used. N.C. indicates negative control (water). (G) Amplicons of the PCR product using Fw #1-Rv #1 primers were subjected to direct sequencing and the 11,575 bp deletion was confirmed in the KO allele. (H) The number of pups born per plug was counted to assess male fertility. Each WT or KO male was mated with three WT females for three months.