Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows.

Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Although zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species. Here we show in bovine fetal fibroblasts that targeting ZFNickases to the endogenous ß-casein (CSN2) locus stimulates lysostaphin gene addition by homology-directed repair. We find that ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete lysostaphin in their milk and in vitro assays demonstrate the milk's ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine.

Developmental expression and possible functional roles of mouse Nlrp4e in preimplantation embryos.

Increasing evidence suggests that some Nlrp genes are crucial for oogenesis, folliculogenesis, and early embryonic development. Nlrp4e is one of seven copies of Nlrp4, which plays a putative role in the reproduction system in mice. Gene duplication is regarded as an important driving force behind the evolution of novel genes with new or altered functions. We investigated the role of Nlrp4e in oocyte and preimplantation embryos by determining its expression profile using quantitative real-time polymerase chain reaction. Nlrp4e mRNA accumulated during oogenesis. Moreover, Nlrp4e transcripts were upregulated during the two-cell stage and then declined sharply and became almost undetectable, which represents a crucial time for major embryonic genome activation in the mouse. Knockdown of Nlrp4e in fertilized eggs using RNA interference resulted in arrested development between the two- and eight-cell stages in a dose-dependent manner. However, targeted inhibition of Nlrp4e in germinal-vesicle-stage oocytes had no phenotypic effects on oocyte maturation. The above experiments were also carried out in parthenogenetic embryos to determine the effects of Nlrp4e in embryos without a paternal genome. The results of this study indicate that Nlrp4e, a maternal-zygotic-effect gene, may not be involved in oocyte maturation but may play a critical role in early embryogenesis.

[Efficient delivery of siRNA into mouse preimplantation embryos by electroporation].

We developed a detailed electroporation method to deliver efficiently siRNA into mouse preimplantation embryos. By introducing Cy3 labeled negative control small interfering RNA (siRNA) into mouse preimplantation embryos, we optimized conditions for the electroporation, including the voltage, pulse duration, pulse number, electroporation buffer and an important step to weaken the zona pellucida. Embryonic survival rate, transfection rate and blastocyst development rate were evaluated under the converted fluorescence microscope, by embryos counting and statistical analysis. The best transfection was achieved in opti-MEM under the conditions of 30 V, 1 ms, 3 pulses, and the duration of digestion in tyrode's solution was 10 s. In conclusion, the proposed electroporation approach here is a simple and efficient tool to deliver siRNA for RNA interference (RNAi) into mouse preimplantation embryos.

Nlrp2, a maternal effect gene required for early embryonic development in the mouse.

Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (∼118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse.