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doxycycline (DOX), achieving an 80% knockout efficiency. Following the administration of polyethylenimine (PEI)-encapsulated plasmids into chicken embryos, we successfully implemented the inducible system in vivo. Quantitative analysis confirmed a mosaic knockout of DMRT1 with an observed efficiency reaching 45%. Following targeted disruption, we evaluated sex-related gene and protein expression alterations via qRT-PCR and Western blot (WB). Furthermore, ELISA was performed to measure testosterone levels in male embryonic gonads across multiple developmental stages (E4.5 to E18.5). qRT-PCR analysis showed that after induction, female-related genes (CYP19A1, FOXL2, ESR1) were significantly upregulated, and male-related genes (DMRT1, SOX9, AMH) were significantly downregulated. WB results revealed increased protein expression levels of CYP19A1 and FOXL2, and decreased protein expression of SOX9 post-induction. ELISA confirmed that testosterone levels in the gonads of induced male embryos were significantly reduced compared to normal and non-induced males. The study successfully established an inducible DMRT1 knockout system in chickens. This system effectively regulates the expression of sex-related genes and reduces testosterone levels in male embryos, providing theoretical and technical support for breeding novel sex-controlled breeding materials.
Liu et al. (Sat,) studied this question.