Effect of aquaporin 3 knockdown by RNA interference on antrum formation in sheep secondary follicles cultured in vitro

SummaryThe objectives were to develop an effective protocol for transfection of ovine secondary follicles and to assess the effect of attenuating aquaporin 3 (AQP3) using a small interfering RNA (siRNA-AQP3) on antrum formation and follicular growth in vitro. Various combinations of Lipofectamine® volumes (0.5, 0.75 or 1.0 µl), fluorescent oligonucleotide (BLOCK-iT ™) concentrations (3.18, 27.12 or 36.16 nM) and exposure times (12, 14, 16, 18 or 20 h) were tested. The BLOCK-iT™ was replaced by siRNA-AQP3 in the transfection complex. Ovine secondary follicles were isolated and cultured in vitro for 6 days using standard protocols. Follicles were transfected on day 0 or 3 or on both days (0 and 3) and then cultured for an additional 3 or 6 days. As revealed by the fluorescence signal, the Lipofectamine®/BLOCK-iT™ complex (0.75 µl + 27.12 nM by 12 h of incubation) crossed the basement membrane and granulosa cell and reached the oocytes. In general, the rate of intact follicles was higher and the rate of antrum formation was lower in transfected follicles compared with control follicles. In conclusion, ovine secondary follicles can be successfully transfected during in vitro culture, and siRNA-mediated attenuation of AQP3 gene reduced antrum formation of secondary follicles.

A Soybean Oil-Based Liposome-Polymer Transfection Complex as a Codelivery System for DNA and Subunit Vaccines

Inexpensive liposome-polymer transfection complexes (LPTCs) were developed and used as for DNA or protein delivery. The particle sizes of the LPTCs were in the range of 212.2 to 312.1 nm, and the zetapotential was +38.7 mV. LPTCs condensed DNA and protected DNA from DNase I digestion and efficiently delivered LPTC/DNA complexes in Balb/3T3 cells. LPTCs also enhanced the cellular uptake of antigen in mouse macrophage cells and stimulated TNF-αrelease in naïve mice splenocytes, both indicating the potential of LPTCs as adjuvants for vaccines.In vivostudies were performed usingH. pylorirelative heat shock protein 60 as an antigen model. The vaccination of BALB/c mice with LPTC-complexed DNA and protein enhanced the humoral immune response. Therefore, we developed a DNA and protein delivery system using LPTCs that is inexpensive, and we successfully applied it to the development of a DNA and subunit vaccine.

EVALUATION OF MAGNETIC-HYDROXYAPATITE NANOPARTICLES FOR GENE DELIVERY CARRIER

With the advancement of nanotechnology, nano-sized bioceramics have been dedicated much attention to biomedical applications in the past decades. In this study, the magnetic-hydroxyapatite nanoparticles (mHap NPs) were synthesized and magnetized by the addition of irons into hydroxyapatite using precipitation process. The mHap NPs were with good biocompatibility and superparamagnetic property which would have great promise in biological applications. We have evaluated the essential properties including plasmid DNA (pDNA) binding capacity by electrophoresis, qualitative transfection efficiency by fluorescence microscopy and quantitative GDNF expression by enzyme-link immunosorbent assay (ELISA). The results indicated that mHap NPs could be successfully binding with pDNA to form pDNA-mHap NPs transfection complex. The endocytosis of pDNA-mHap NPs transfection complex was mediated into cells and facilitated if magnetic field was applied. The transfection efficiency could also be significantly improved by magnetofection. The gene expression had been four-fold increase in high pDNA loading (2 μg/well) in transfection procedure with magnetic field compared with traditional transfection method. In brief, the mHap NPs have great potential as non-viral inorganic delivery carriers for gene delivery system.