Fig. 1.

Expression and knockout of Selenov and the knockout effects on reproduction and body weight of mice. All mice were male, fed a normal diet (Se-adequate), and 4-month old unless otherwise indicated. (A) Expression of Selenov mRNA in various tissues of WT mice (n = 5) relative to that of testis (set as 100). (B) Expression of Selenov mRNA in testis of male WT mice at ages of 3 months (3 m) and 1 year (1 y) relative to that at age of 3 weeks (3 w, set as 10) (n = 5, bars showing a different letter were different at P < 0.05). (C) CRISPR/Cas9 gene-targeting strategy for the generation of Selenov KO mice. Two sgRNAs (black triangles) were designed to generate a ~3 kb deletion covering the entire coding region of Selenov. Primers F1 and R2 amplified an 808 base-pair (bp) product in the targeted allele; primers F1 and R1 amplified a 675 bp product in the WT allele. (D) Genotyping of the Selenov WT (+/+) and KO (−/−) by PCR of genomic DNA: WT (+/+) had a 675 bp band, KO (−/−) had an 808 bp band, and heterozygote (±) had both bands. (E) RNAscope images (in-situ hybridization) showing high and low expression of Selenov in the testis and EWAT of WT mice, respectively (red arrows indicating the presence of Selenov), but no detectable expression in either tissue of the KO mice. Scale bars, 100 μm for testis and 50 μm for EWAT. The image was a representative of five sets of data. (F) Western blot analysis of SELENOV in the testis of WT and KO mice (n = 3). (G) Images of sperm (upper panel, magnification, × 8) and H&E staining of testis (bottom panel, scale bar, 100 μm) showing no apparent morphological abnormalities by the KO. The image was a representative of five sets of data. (H) Average body weights of mice at 4, 5, and 6 months of age (n = 10): *P < 0.05 for the body difference between the WT and KO mice. Data in (A), (B), and (H) are mean ± SE. Abbreviations: BAT, brown adipose tissue; bp, base pairs; EWAT, epididymis white adipose tissue; WAT, white adipose tissue; KO, knockout; WT, wild-type. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)