Pronuclear microinjection is not suitable for RNA polymerase III promoter driven constitutive RNAi transgenesis in mice for XY male-to-female sex reversal by <i>Sry</i> gene knockdown

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. <i>Sry</i> (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of <i>Sry</i> 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 <i>Sry</i> gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting <i>Sry</i> 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