Our lab has constructed a new nonviral vec-tor—hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hF Ⅷ (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII- BDDAK39 in the hrDNA locus. The hFⅧ-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific inte-gration was 2.0×10?5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells?1·24 h?1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.
LIU Xionghao LIU Mujun SHE Hua WEN Lu XUE Zhigang LIANG Desheng CAI Fang PAN Qian LONG Zhigao WU Lingqian DAI Heping XIA Kun XlA Jiahui
In order to develop a safe and effective gene therapy carrier, some toxicological and biodynamical ex- periments were carried out on silica nanoparticles (SiNPs). First we prepared SiNPs with appropriate portions of cyclo- hexane, deionized water and ethyl silicate, and then trans- fected the modified SiNPs and GFP plasmid DNA complex into the HT1080 cells to test the effectiveness of transfection for gene therapy. At the same time, we injected the SiNPs into a number of mice through tail vein. Then we made the mice crossed to evaluate the acute, long-term and reproduc- tive toxicity. In vivo distribution analysis and pathological examination were made on both adult mice and their off- spring. SiNPs were uniform and had an average diameter of 40 nm, and the modified SiNPs carried exogenous DNA molecules into target cells and the transferred GFP fusion gene was effectively expressed in the cells. The SiNPs injected via tail vein were widely distributed in almost all of tissues, and the injected mice had the ability to reproduce normally. The in vivo and in vitro results of this study clearly show that SiNPs can be used as a safe and effective carrier for gene transfection and gene therapy.
