Short hairpin RNA (shRNA)-mediated gene regulation is a commonly used technique for gene manipulation. An efficient and safe delivery system is indispensable when shRNA is delivered into living organisms for gene therapy. Previous studies have proved that DNA and protein can be delivered into dendritic cells (DCs) by non-pathogenic Saccharomyces cerevisiae without being degraded. CD40 is closely related to apoptosis of tumor cells and some immune mechanisms. In this study, we demonstrated that recombinant yeast S. cerevisiae efficiently delivered the shRNA of immune-associated gene (CD40) into mouse intestinal DCs via oral administration. Western blot analysis of isolated intestinal DCs indicated that the inhibition of CD40 gene expression reached up to 56–91%. The secretion of cytokines such as interleukin-2 (IL-2), IL-6, IL-10, IL-12, tumor necrosis factor-α and interferon-γ in intestinal DCs had varying degrees of changes. In conclusion, we found that orally administered recombinant yeast can be used as an efficient shRNA delivery system for intestinal DC-specific gene silencing and immunomodulation in vivo.
Transforming growth factor-β (TGFβ) superfamily are critical regulators of germ cell development that act as extracellular ligands of the signal transduction pathways regulating proliferation, apoptosis and other aspects of cell behaviour. As a member of the TGF-β superfamily, growth differentiation factor 9 (GDF9) plays a critical role in ovarian follicular development and the ovulation rate in females; however, its role in the testis has not been well elucidated. The aim of this study was to investigate the expression of GDF9 and its receptor genes BMPRII and ALK5 in prepuberal bovine Sertoli cells (SCs). In addition, we assessed the effects of GDF9 on immature SCs apoptosis, the cell cycle and tight junction functions. We found that GDF9 and its receptor genes BMPRII and ALK5 were expressed in immature SCs. Exogenous GDF9 significantly promoted SCs proliferation and inhibited the apoptosis of SCs by significantly upregulating Cyclin E (cell cycle) and bcl-2 (anti-apoptosis) mRNA expression and downregulating caspase-3 (pro-apoptosis) mRNA expression. Meanwhile, exogenous GDF9 significantly decreased the value of transepithelial electrical resistance by significantly downregulating claudin-11 mRNA expression and influencing the distribution of occludin. In conclusion, this study reveals that GDF9 is a key regulator of bovine SCs through the modulation of the cell cycle, apoptosis and tight junction functions.
CCAAT/enhancer-binding protein beta (C/EBPβ), an important transcriptional factor, plays a pivotal role in the regulation of female germ cell development. However, the role of C/EBPβ on the development of male germ cells has not been reported. In this study, we constructed the recombinant adenovirus plasmids of bovine C/EBPβ gene and harvested the subsequent adenoviruses, and then assessed the mRNA levels of spermatogenesis-related genes (real-time PCR) and secretion of inhibin B after 48 h of Ad-C/EBPβ recombinant adenovirus infection in bovine sertoli cells (SCs). We found that overexpression of exogenous C/EBPβ gene upregulated the mRNA expressions of spermatogenesis-related genes, including Pdgfa, Claudin, Caspase-3, Occludin, kit1 and Cyclin E, and decreased the mRNA levels of Cyclin D1. Meanwhile, overexpression of exogenous C/EBPβ gene significantly increases the amounts of secreted inhibin B. In conclusion, the results indicate that the C/EBPβ gene plays an important regulatory role in regulation of the cell cycle regulators and spermatogenesis-related genes expression and function of bovine SCs.
With an aim to improve the efficiency of yak-bovine interspecies somatic cell nuclear transfer (iSCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak-bovine iSCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (mSOF) or relative defined culture medium (RDCM) with 5% or 20% oxygen tension. Relative mRNA abundance of Oct-4, IFNT, IGF-2, Bax, GPX-1, SOD-1, CAT and GSS was analysed in blastocysts with qRT-PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension (P < 0.05). The total cell number of blastocyst derived from RDCM with 20% oxygen tension was lower than that of other groups, whereas the group of RDCM with 5% oxygen tension showed a beneficial effect on apoptosis index and tolerance to cryopreservation (P < 0.05). However, under the same oxygen tension, the mRNA abundance of IFNT of RDCM groups was higher than that of the mSOF groups. In addition, high oxygen tension during in vitro culture (IVC) with RDCM significantly increases the mRNA expression of oxidative stress-related genes (GPX-1, SOD-1, CAT and GSS) (P < 0.05). 3, 4-Dihydroxyflavone (DHF) during high oxygen tension was able to improve the cloned blastocyst formation rate in RDCM (P < 0.05). These results for the first time showed that low oxygen tension and RDCM could improve the developmental competence and quality and alleviate the oxidative stress for yak-bovine iSCNT embryo during IVC.
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