Construction of expression vectors using initiation sites of DNA replication and their application to gene therapy

利用DNA复制起始位点构建表达载体及其在基因治疗中的应用

基本信息

批准号：
04557105
负责人：
ARIGA Hiroyoshi
金额：
$ 9.28万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

We have determined the initiation regions in the various genes including human c-myc, N-myc, p53, hsp70, and mouse immunoglobulin heavy chain gene (IgH). The plasmid clones containing the these origin regions were also functioning as the ARSs in transfected mammalian cultured cells and transgenic mouse cells. These systems should be useful for the expression vector to produce the bio-active reagents in a large amounts. Furthermore, we have identified the protein factors that regulate the replication levels of the introduced plasmid DNAs. The combination between the DNA element essential for the initiation of DNA replication and protein factors regulating the replication enhances the production of the materials of interest. Details we have clarified are following.HindIII-PstI region positioned upstream of c-myc gene was identified as origin and transcriptional enhancer. Clone containing this region replicated in cltured cells and transgenic mice in an episomal state. The 21 nucleotide w … More as identified to be essential for replication. The protein complex containing c-myc protein was then identified to act the regulation of this plasmid DNA.One of the protein involved in this c-myc protein complex was identified as MSSP, and two cDNAs were cloned, MSSP-1 and MSSP-2. These proteins bound to both double and single stranded DNA in a sequence-specific manner, and promoted the initiation of replication. One of the plasmid recovered from transgenic mice bearing this c-myc-origin possessed the extra sequence beside the c-myc origin. This sequence, TRM, enhanced the efficiency of replication to approximately 100 fold to that without TRM.Binding proteins recognizing TRM were next identified.We further identified the origins in the various genes described above. Essential sequences and recognition proteins to function were next determined as in the case of c-myc gene. Detail experiments were carried out, and we got the similar relationship between cis and trans elements.The experimental results described here show the promising strategy for application to the gene therapy. Less

我们已经确定了各种基因中的主动区域，包括人C-Myc，N-MYC，p53，HSP70和小鼠免疫球蛋白重链基因（IGH）。包含这些原点区域的质粒克隆在翻译的哺乳动物培养细胞和转基因小鼠细胞中也起作用。这些系统对于表达载体应大量产生生物活性试剂有用。此外，我们已经确定了调节引入质粒DNA的复制水平的蛋白质因子。 DNA元件对启动DNA复制至关重要的组合之间的组合与重复复制的蛋白质因子之间的结合增强了感兴趣材料的产生。我们已经阐明的细节正在遵循。将C-MYC基因上游定位的Hindiiii-PSTI区域鉴定为原点和转录增强子。含有该区域的克隆在偶发状态下复制的细胞和转基因小鼠。 21核苷酸W…更多地确定为复制至关重要。然后确定含有C-MYC蛋白的蛋白质复合物以作用于该质粒DNA的调节。将参与该C-MYC蛋白复合物涉及的蛋白质的一种被鉴定为MSSP，并克隆了两个CDNA，MSSP-1和MSSP-2。这些蛋白质以序列特异性方式与双重和单链DNA结合，并促进了复制的主动性。从带有这种C-Myc-基的转基因小鼠中回收的质粒之一，在C-Myc起源旁边具有额外的序列。接下来鉴定了该序列TRM，将复制的效率提高到大约100倍的情况下，而没有Trm。结合蛋白质识别TRM。我们进一步鉴定了上述各种基因中的起源。接下来确定了起作用的基本序列和识别蛋白，如C-MYC基因而言。进行了详细的实验，我们得到了顺式和反式元素之间的相似关系。此处描述的实验结果显示了适用于基因治疗的有望策略。较少的