In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate.

INTRODUCTION: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. MATERIAL AND METHODS: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. RESULTS: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. CONCLUSION: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Cellular Localization Analysis of Enhanced Green Fluorescent Protein Tagged hnRNP A1 Under Stress / 天津医药

Objective To construct eukaryotic enhanced green fluorescent protein (EGFP) expressing recombinant plasmid, pEGFP-C1-hnRNP A1, which contains coding sequence of human hnRNP A1 (heterogeneous nuclear ribonucleo-protein A1), and to perform cellular localization analysis of EGFP tagged hnRNP A1 under stress. Methods Total RNA was isolated from HeLa cell used for synthesis of first-strand cDNAs using reverse primers that are specific for the 3′-un-translated region of hnRNP A1. hnRNP A1 gene fragments were then amplified by touch-down PCR from those cDNAs and inserted into pEGFP-C1 fluorescent bearing vector through EcoRⅠ/BamHⅡdouble enzyme digestion and T4 DNA Ligase connection. The recombinant pEGFP-C1-hnRNP A1 plasmid was transfected into HeLa cells and green fluorescent tagged fusion proteins was examined by Western blot and confocal fluorescence microscopy. Co-localization of EGFP-hnRNP A 1 with poly (A)+mRNA (the marker of the stress granules), or DCP1a (the marker of processome) were detected by RNA fluores-cence in situ hybridization and immunofluorescence. Results The pEGFP-C1-hnRNP A1 was sequenced and digested cor-rectly by restriction single/double enzyme. The green fluorescent fusion protein was also detected in transfected HeLa cell by Western blot and confocal fluorescence microscopy. EGFP-hnRNP A1 co-localizes with poly(A)+mRNA, but not DCP1a. Conclusion Recombinant eukaryotic plasmid of pEGFP-C1-hnRNP A1 was constructed successfully and expressed effec-tively. EGFP tagged hnRNP A1 takes part in forming stress granules.

Functional study of dextran-graft-poly((2-dimethyl amino)ethyl methacrylate) gene delivery vector for tumor therapy.

The obstacle of gene therapy is the shortage of efficient delivery system. The development of the gene delivery system with high transfection efficiency and low toxicity appears to be crucial. Recently, we reported that the dextran-graft-poly((2-dimethyl amino)ethyl methacrylate) (DPD) can be potentially used as efficient gene vector. Herein, DPD was systematically studied for its potential in tumor gene therapy. DPD was synthesized and characterized by agarose gel electrophoresis, particle size and zeta potential. The particle size and zeta potential of the DPD/enhanced green fluorescent protein (pEGFP-C1) plasmid complexes at various N/P ratios were 130-150 nm and about 40 mV, respectively. The results showed that DPD exhibit a higher transfection effect compared with Lipofectamine 2K (Lipo 2K), a commercialized vector. The possibility of DPD in gene therapy was evaluated using p53, a gene that has been wildly applied in the research of cancer gene therapy. DPD/pEGFP-C1-p53 complex was found to be able to inhibit tumor cell proliferation through cell cycle arrest and apoptosis. Moreover, the tumor growth was found to be restrained when DPD/pEGFP-C1-p53 complex was used in a xenograft MCF7 tumor model in vivo. These observations indicated that DPD/pEGFP-C1-p53 complex may be considered to be an efficient delivery system for tumor gene therapy.

Bone response to the multilayer BMP-2 gene coated porous titanium implant surface.

OBJECTIVES: Evaluate hBMP-2 expression following gene delivery from plasmid multilayers formed on sandblasted titanium in vitro and bone formation around similarly prepared implant surfaces in vivo. MATERIALS AND METHODS: Multilayers of cationic lipid/rhBMP-2 plasmid DNA complex (LDc) and anionic hyaluronic acid (HA) was assembled on sandblasted-dual acid etched pure titanium disks or implant surfaces using layer-by-layer (LBL) assembly. Gene delivery and hBMP-2 expression in cells exposed to the LDc multilayers was measured in vitro. To determine the effect of BMP delivery from such multilyaers in vivo, roughened implants coated with BMP-2 LDc multilayers or uncoated control implants (n = 15 for both) were implanted in the femurs of NZW rabbits. After 2, 4, 8 weeks, femurs were retrieved and prepared for histomorphometric evaluation (n = 5 rabbits per time point). RESULTS: MC3T3-E1 cells cultured directly on the BMP-2 LDc coated titanium disks showed EGFP and hBMP-2 expression after 48 h in culture. Increased gene delivery occurred by increasing the number of assembly layers when cells were cultured for 48 h. Cells cultured on LDc coated surfaces had significantly higher cell viability than control cells cultured on uncoated porous titanium surfaces. Histologic observation of the implants showed that after 4 weeks healing, the bone to implant contact (BIC) on the LDc coated surface was much lower than that on the control surface, but didn't reach significant. In contrast, the percentage of bone within the implant's threads was significantly higher than the control group (P = 0.047). CONCLUSION: The BMP-2 gene coated sandblasted dual acid etched titanium implants slightly accelerated early bone formation around implants.

Construction of eukaryotic expression vectorinsertedby MafA and its expression in HepG-2 cells / 重庆医科大学学报

Objective:To construct eukaryotic expression vectors pEGFP-N2/MafA and pEGFP-C1/MafA,encoding the mouse musculus v-maf musculoaponeurotic fibrosarcoma oncogene family,protein A(MafA)gene,for the further research of its expression in HepG-2 cells.Methods:pRNA of the mouse MafA gene was distilled by RT-PCR from the mouse,and inserted into Hind Ⅲand SalⅠrestriction sites by PCR,then cloned into pEGFP-N2 and pEGFP-C1 vectors to obtain plasmids pEGFP-N2/MafA and pEGFP-C1/MafA.Human liver cancer cells(HepG-2) were transfected with formed plasmids by means of lipidosome.The MafA-EGFP fused protein was viewed directly with fluorensce microscope,and expression of MafA and insulin Ⅱ was detected by RT-PCR.Results:The mouse MafA gene was amplified through RT-PCR and successfully cloned into transfer vectors.The favorite gene sequence could be expressed and was completely consistent with that reported in genebank,but the insulin Ⅱ gene expression was not detected.Conclusion:The recombinant plasmids pEGFP-N2/MafA and pEGFP-C1/MafA were successfully constructed.But the insulin Ⅱgene was not expressed.