No abstract

The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

No abstract

Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.

The mesoscale atmospheric environment of a dust event in the Tarim Basin, Taklimakan Desert, one of the key sources of aeolian dust in Northeast Asia, is investigated. Dust events in the Taklimakan Desert, including blowing dust and suspended dust, have long-lasting attributes, whereas in the Gobi Desert, dust events occur in phase with the passage of synoptic cyclones. Numerical simulations are conducted for a well-defined dust event on 12-15 April 2002. The Regional Climate Model of the Meteorological Research Institute (MRI-RCM) is employed. The MRI-RCM horizontal grid size is 20 km. The model reasonably well simulates time variations and spatial distributions of the surface wind field. Three types of mesoscale flow are revealed by the simulation. Sequential formation and/or coexistence of these flows can cause wind intensification and active dust emission from various areas across the basin. This gives a possible explanation for the long lasting dust events in the Tarim Basin. A closer examination shows that the development of these mesoscale circulations is closely related to the large-scale flow field behind the surface low-pressure system.

The site-unspecific method could successfully identify most of the peptides from tryptic hydrolysates revealed by site-specific identification.

Renal interstitial fibrosis is a necessary step in the progression of chronic kidney to end stage renal disease. MicroRNA-29 (miR-29) has been shown to play essential roles in epithelial-mesenchymal transition (EMT), and thus may contribute to the regulation of renal interstitial fibrosis. However, the role of miR-29 in the regulation of EMT during chronic kidney disease and renal transplantation has been a source of intense debate, and the mechanisms underlying this process are incompletely understood. In this study, we investigated the function of miR-29b in the regulation of EMT and to gain a better understanding of the mechanism by which miR-29b modulates EMT by targeting the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway during the process of renal interstitial fibrosis. The rat proximal tubular epithelial cell line NRK-52E was cultured in DMEM and treated with angiotensin II (Ang II) at various concentrations. RT-PCR was performed to investigate changes in the levels of expression of miR-29b in NRK-52E cells and western blotting was used to analyze the expression of PI3K, p-AKT, vimentin and keratin 18. The result of the study show that treatment of NRK-52E cells with Ang II induced the transition of the cellular phenotype from epithelial to mesenchymal and upregulated the PI3K/AKT signaling pathway; this was also found following treatment with a phosphatase and tensin homolog on chromosome 10 (PTEN)-specific inhibitor. Increased expression of miR-29b was able to reverse the phenotype induced by Ang II in NRK-52E cells and blocking miR-29b activity with an miR-29b inhibitor resulted in enhanced EMT. Additionally, the PI3K/AKT signaling pathway was found to be suppressed in the presence of enhanced expression of miR-29b by direct binding to 3'-untranslated region (3'-UTR) of PIK3R2. We concluded that miR-29b plays an important role in the negative regulation of Ang II-induced EMT via PI3K/AKT signaling pathway and propose that enhancing miR-29b level or blocking PI3K/AKT signaling pathway may be a novel therapeutic target in renal interstitial fibrosis.

The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.