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

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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