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

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)。含有这些起始区的克隆在转基因哺乳动物培养细胞和转基因小鼠细胞中也具有ARS的功能。这些系统将有助于表达载体大量生产生物活性试剂。此外，我们还鉴定了调节导入的质粒DNA复制水平的蛋白质因子。启动DNA复制所必需的DNA元件和调节复制的蛋白质因子之间的结合提高了感兴趣材料的产量。我们已明确的细节如下：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。含有该区域的克隆在克隆细胞和处于异体状态的转基因小鼠中复制。21个核苷酸w…更多被确定为对复制至关重要的内容。鉴定了含有c-myc蛋白的蛋白质复合体对该质粒DNA的调控作用，其中一个涉及c-myc蛋白复合体的蛋白质被鉴定为MSSP，并克隆了两个cDNA：MSSP-1和MSSP-2。这些蛋白质以序列特异性的方式与双链和单链DNA结合，并促进复制的启动。从带有该c-myc起源的转基因小鼠中回收的一个质粒，除了c-myc起源之外，还含有额外的序列。这个序列TRM将复制效率提高了大约100倍，识别TRM的结合蛋白被进一步鉴定。我们进一步鉴定了上述各种基因的起源。接下来，与c-myc基因的情况一样，确定起作用的基本序列和识别蛋白。我们进行了详细的实验，得到了顺式和反式元素之间的相似关系，为基因治疗的应用提供了有前景的策略。较少

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