Figure 5.

Generation of Start-KO mice and morphological characterization of Start-KO at 2.5 months. (A) Strategy for deletion at the Start locus. Start is a transcript of 1822 nucleotides in length as shown by a black box. Positions of target sequences of gRNAs are indicated by dashed lines with scissor pictures. By injection of these gRNAs and CAS9 protein into fertilized eggs, two Start-KO lines, #1 and #2, were established. The deleted regions of the two KO lines are indicated by the number relative to the transcriptional start site of Start. (B) A representative result of genomic PCR for genotyping. Genome DNAs from wild-type and Start-KO #1 and #2 mice were subjected to PCR amplifying a 4021-bp region including the deleted region. The upper band corresponds to the wild-type locus and the lower band indicates the deleted locus. (C) A representative result of RT-PCR for Start expression. Total RNAs were collected from adult wild-type and Start-KO testes (lines #1 and #2) and used for RT-PCR. The Gapdh gene was detected as an internal control. The cycle number of each PCR is shown in parenthesis. (D) Ratio of testis to body weight at 2.5 months (lines #1 and #2). Data are presented as means ± S.D. from four independent sets of wild-type and Start-KO littermates (four biological replicates). Student’s t-test revealed no significant difference between wild-type and KO mice. (E) Testis section at 2.5 months (line #2). Testes from 2.5-month-old wild-type and Start-KO littermates were fixed with Bouin’s solution, dehydrated, embedded in paraffin, cut into 7-µm-thick sections, and stained with hematoxylin and eosin. No difference was observed. Scale bars, 100 µm. (F) Sperm number at 2.5 months (line #2). Mature sperms were isolated from the cauda epididymis of three sets of 2.5-month-old wild-type and Start-KO littermates in HTF medium (three biological replicates). After fixation with 5% formalin, the number was counted. Data are presented as means ± S.D. Student’s t-test showed no significant difference.