Establishment of gene therapy vector by using DNA replication origin and virus vector

利用DNA复制起点和病毒载体建立基因治疗载体

• 批准号: 07557147
• 负责人: ARIGA Hiroyoshi
• 金额: $ 8.7万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557147/
• 关键词: MYC; adenovirus; vector; heat shock protein; transcription; DNA replication; c-myc; DNA複製開始; 結合タンパク質; 癌遺伝子; DNA結合; 転写因子; 発現ベクター; 細胞周期

Expression vector composed of adenovirus, beta-galactosidase, and DNA replication origin (ori) was constricted.Oris used here were from genes of c-myc, human hsp70 and immunoglobulin heavy chain in addition to SV40 origin as a positive control. These origins were inserted to adenovirus vector containing Cre-lox system, and the recombinant adenovirus were recovered from human 2983 cells transfected with these vectors. Adenovirus containing SV40 origin were first infected to monkey CosI cells, and the circular adenovirus expressing beta-galactosidase was recovered. Adenovirus containing hsp70 gene origin were then infected to human HeLa cells, and the circular form of adenovirus DNA was also recovered. Copy number of hsp70 ori derived plasmid DNA in the cells, however, was lower than that of SV40 origin in 10^<-3> order. Results suggest that this system should be useful for an expression vector for gene therapy.

构建了由腺病毒、β-半乳糖苷酶和DNA复制起点（ori）组成的表达载体，所用的ori分别来自c-myc、人hsp 70和免疫球蛋白重链基因，并以SV 40为阳性对照。将重组腺病毒插入到含有Cre-lox系统的腺病毒载体中，并从用这些载体转染的人2983细胞中回收重组腺病毒。首先将含有SV 40来源的腺病毒感染猴CosI细胞，回收表达β-半乳糖苷酶的环状腺病毒。然后将含有hsp 70基因来源的腺病毒感染人HeLa细胞，也回收了环状形式的腺病毒DNA。然而，细胞中hsp 70 ori来源的质粒DNA的拷贝数比SV 40来源的拷贝数低10个数量<-3>级。结果表明，该系统应该是有用的表达载体的基因治疗。

项目成果

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Taira, T.：“在人类热休克蛋白中鉴定出一种新型 G1/S 特异性增强子”Nucleic Acids Res.25。

Taira,T.: "A novel G1/S-specific enhancer identified in the human heat shock protein 70 Gene" Nucleic Acids Res.25. 1975-1983 (1997)

Taira,T.：“在人类热休克蛋白 70 基因中鉴定出一种新型 G1/S 特异性增强子”Nucleic Acids Res.25。

Manabe,A.: "Transcriptional repression activity of N-myc protein requires phosphorylation by MAP kinase." Biochim Biophys.Res.Commun.in press.

Manabe,A.：“N-myc 蛋白的转录抑制活性需要 MAP 激酶磷酸化。”

Manabe, A.: "Transcriptional repression activity of N-myc protein requires phosphorylation by MAP kinase." Biochem. Biophys. Res. Comm.219. 813-823 (1996)

Manabe, A.：“N-myc 蛋白的转录抑制活性需要 MAP 激酶的磷酸化。”

Izumi,S.: "Molecular cloning of the complementary DNA for the mouse pyruvate kinase M-2 gene whose expression is dependent upon cell differentiation." Biochim.Biophys.Acta. 1267. 135-138 (1995)

Izumi,S.：“小鼠丙酮酸激酶 M-2 基因互补 DNA 的分子克隆，该基因的表达依赖于细胞分化。”