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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.
Our lab has constructed a new nonviral vec-tor-hr DNA 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 Southern blotting, and tested the expression of hFVIII- BDDAK39 in the hr DNA locus. hFVIII-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific interation was 2.0 x 10? 5. hFVIII-BDDAK39 in hrDNA locus of HT-1080 was found to be capable to express efficiently (32 ± 5 ng · 106 cells · 1 · 24 h · 1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.