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| Expression of reconstructive hFⅧ in the hrDNA by using hrDNA targeting vector | |
| 引用本文： | 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. Expression of reconstructive hFⅧ in the hrDNA by using hrDNA targeting vector[J]. 科学通报(英文版), 2005, 50(19): 2187-2192. DOI: 10.1007/BF03182670 |
| 作者姓名： | 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 |
| 作者单位： | National Lab of Medical Genetics of China, Central South University,Changsha 410078, China |
| 基金项目： | This work was supported by Chinese 973 Projects （Grant No. 2004CB518800）, 863 Projects （Grant Nos. 2002BA711A07-08, 2002BA711A07-03 ＆ 2002AA227011）, the National Natural Science Foundation of China （Grant No. 31830200） and Life Science Research Foundation of Hunan Province. |
| 摘 要： | Our lab has constructed a new nonviral vector-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 integration was 2.0×10^-5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently （32±5 ng·10^6 cells^-1 .24 h^-1）. Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia. |
| 关 键 词： | hrDNA 目标向量 人类起源 遗传表达 核糖体 器官重组 |
| 收稿时间： | 2005-06-21 |
| 修稿时间： | 2005-06-212005-07-25 |
Expression of reconstructive hFVIII in the hrDNA by using hrDNA targeting vector |
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| Xionghao Liu,Mujun Liu,She Hua,Wen Lu,Zhigang Xue,Desheng Liang,Fang Cai,Qian Pan,Zhigao Long,Lingqian Wu,Heping Dai,Kun Xia,Jiahui Xia. Expression of reconstructive hFVIII in the hrDNA by using hrDNA targeting vector[J]. Chinese science bulletin, 2005, 50(19): 2187-2192. DOI: 10.1007/BF03182670 | |
| Authors: | Xionghao Liu Mujun Liu She Hua Wen Lu Zhigang Xue Desheng Liang Fang Cai Qian Pan Zhigao Long Lingqian Wu Heping Dai Kun Xia Jiahui Xia |
| Affiliation: | 1. National Lab of Medical Genetics of China, Central South University, 410078, Changsha, China |
| Abstract: | Our lab has constructed a new nonviral vector—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 hFVIII (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 integration 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. |
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