Fig. 2

Generation of Spindoc knockout mouse models. (A) Schematic diagram shows the targeting strategy for generating Spindoc knockout (KO) mice using the CRISPR/Cas9 system. Blue boxes represent exons of the Spindoc gene on mouse chromosome 19. The genomic sequence targeted by a pair of sgRNAs are underlined with PAM sequences being highlighted in purple color. The red arrowheads point to the cut sites around exon 2 of Spindoc gene. Two F0 founder lines were generated - Line 1 carried 1 nt (T) insertion, while Line 2 carried a combination of 22 bp deletions as indicated; (B, C) Sanger sequencing of genomic DNA from mouse tail clips showing the frameshift variant (+ 1 nt) of KO mice (Line 1) (B), and the other frameshift variant (− 22 nt) (Line 2) (C); (D) Representative western blot analyses validated the Spindoc protein levels in WT and KO adult testes. GAPDH was used as a loading control. (E) Quantitative analysis of Spindoc protein levels in WT and KO adult testes. Data are presented as mean ± SEM, n = 3. P < 0.001 by student t-test. (F) Representative morphology of testes and epididymis from WT, Heterozygote (HET) and KO mice. The testis of KO was smaller than that of the WT; the epididymis of KO was more transparent than that of the WT. (G) Histogram showing the testis weights in WT and KO adult mice. Data are presented as mean ± SEM, n = 3. p < 0.001 by student t-test