Abstract
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.
Highlights
RNA interference (RNAi) has become a routine genetic tool to study gene function in mammalian cells including cancer cells [1]
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
Male-to-female sex reversal could not occur with the polIII promoter driven constitutive short hairpin RNA (shRNA) expressing transgene by pronuclear microinjection method
Summary
RNA interference (RNAi) has become a routine genetic tool to study gene function in mammalian cells including cancer cells [1]. Short hairpin RNA (shRNA) expression vectors have been developed which can be designed for knockdown of specific gene expression [2]. The standard methods of shRNA transgenic (Tg) mice production have not been established. ShRNA targeting Sry was constructed [7] and the corresponding Tg mice were attempted to be generated. As the pronuclear microinjection method is standard method for production of Tg mice, shRNA Tg mice were produced by pronuclear microinjection method in this study. ShRNA targeting Sry gene will be useful tool to establish the methods of RNAi transgenesis in mice
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