Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coi...Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. The importance of the strict regulation of Sry expression remains unknown. Thus, we attempted to produce a Sry ubiquitous-expressing transgenic (Tg) mouse in which foreign Sry is driven by the CAG (cytomegalovirus immediate-early enhancer, chicken beta-actin promoter, and the fusion intron of chicken beta-actin and rabbit beta-globin)-Sry gene for ubiquitous expressing Sry. A low rate (2/127) of Tg pups was observed, whereas the rate of early-stage transgenic embryos before birth was 19.2% (5/26). The Sry ubiquitous-expressing embryos showed abnormal development. The results suggest that ubiquitous expression of Sry exerts a negative effect on embryonic development. One of the two adult Tg mice showed low levels of Sry expression. The other Tg mouse showed high Sry transgene expression, but was mosaic for the transgene. Developmental analysis of transgenic F1 embryos produced from the mosaic Tg mouse revealed that ubiquitous expression of Sry had a lethal effect on embryonic development around 12.5 dpc. The histological data indicated that ubiquitous expression of Sry induced abnormal cardiovascular development, resulting in embryonic death. Enhanced expression of Sry suppressed endogenous Tie2/Tek (tyrosine kinase with Ig and EGF homology domains 2/tunica interna endothelial cell kinase) expression in Sry-transfected primary cultured cells from wild type embryonic hearts. The results indicate that the tissue-specific and stage-specific expression of Sry is essential for normal embryogenesis.展开更多
Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard met...Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.展开更多
文摘Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. The importance of the strict regulation of Sry expression remains unknown. Thus, we attempted to produce a Sry ubiquitous-expressing transgenic (Tg) mouse in which foreign Sry is driven by the CAG (cytomegalovirus immediate-early enhancer, chicken beta-actin promoter, and the fusion intron of chicken beta-actin and rabbit beta-globin)-Sry gene for ubiquitous expressing Sry. A low rate (2/127) of Tg pups was observed, whereas the rate of early-stage transgenic embryos before birth was 19.2% (5/26). The Sry ubiquitous-expressing embryos showed abnormal development. The results suggest that ubiquitous expression of Sry exerts a negative effect on embryonic development. One of the two adult Tg mice showed low levels of Sry expression. The other Tg mouse showed high Sry transgene expression, but was mosaic for the transgene. Developmental analysis of transgenic F1 embryos produced from the mosaic Tg mouse revealed that ubiquitous expression of Sry had a lethal effect on embryonic development around 12.5 dpc. The histological data indicated that ubiquitous expression of Sry induced abnormal cardiovascular development, resulting in embryonic death. Enhanced expression of Sry suppressed endogenous Tie2/Tek (tyrosine kinase with Ig and EGF homology domains 2/tunica interna endothelial cell kinase) expression in Sry-transfected primary cultured cells from wild type embryonic hearts. The results indicate that the tissue-specific and stage-specific expression of Sry is essential for normal embryogenesis.
文摘Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.
