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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的起始区，包括人 c-myc、N-myc、p53、hsp70 和小鼠免疫球蛋白重链基因 (IgH)。含有这些起源区域的质粒克隆也在转染的哺乳动物培养细胞和转基因小鼠细胞中充当ARS。这些系统应该可用于表达载体大量生产生物活性试剂。此外，我们还确定了调节引入的质粒 DNA 复制水平的蛋白质因子。启动 DNA 复制所必需的 DNA 元件与调节复制的蛋白质因子之间的结合增强了目标材料的产生。我们已经澄清的细节如下。位于c-myc基因上游的HindIII-PstI区域被鉴定为起源和转录增强子。含有该区域的克隆以游离状态在培养细胞和转基因小鼠中复制。 21 个核苷酸被确定为复制所必需的。然后鉴定出含有c-myc蛋白的蛋白复合物对该质粒DNA进行调节。该c-myc蛋白复合物中涉及的蛋白之一被鉴定为MSSP，并且克隆了两个cDNA：MSSP-1和MSSP-2。这些蛋白质以序列特异性方式结合双链和单链 DNA，并促进复制的启动。从携带该c-myc-起源的转基因小鼠中回收的质粒之一除了c-myc-起源之外还具有额外的序列。该序列TRM将复制效率比没有TRM的复制效率提高了大约100倍。接下来鉴定了识别TRM的结合蛋白。我们进一步鉴定了上述各种基因的起源。接下来确定发挥功能的必需序列和识别蛋白，就像c-myc 基因的情况一样。进行了详细的实验，我们得到了顺式和反式元件之间的相似关系。这里描述的实验结果显示了应用于基因治疗的有前途的策略。较少的