Construction of high-expression vector had on gene-amplified chromosomal location and its application to human cell

基因扩增是指“基因组中某个或多个基因的选择性重复复制，而其他基因没有成比例增加”。基因扩增是真核生物中普遍存在的现象。它是许多生物发育的重要过程，是肿瘤细胞和一些人类寄生虫产生耐药性和肿瘤成熟的重要过程。从20世纪80年代初开始，这种基因扩增被用于利用中国仓鼠卵巢（CHO）细胞生产治疗性蛋白的工业生产。在哺乳动物细胞中生产这种蛋白质最紧迫的问题是重组蛋白的低生产率。为了提高生产力，转染基因的基因扩增细胞工程技术被广泛应用，其中可扩增基因被用作转染载体的选择标记。首先将含有目标蛋白cDNA和可扩增标记基因的载体导入宿主细胞系。当可选择和可扩增的基因被扩增时，含有目标cDNA的遗传连锁序列被共扩增。因此，转染的DNA可以很容易地获得高水平的表达。本研究通过构建CHO细菌人工染色体文库来分析基因扩增机制。我们确定了基因扩增位置的结构，并估计了基因扩增的机制。

Gene amplification means "the selective, repeated replication of a certain gene or genes without a proportional increase in other genes in the genome". Gene amplification is a widespread phenomenon in eukaryotes. It is an important process in the development of many organisms, the emergence of drug resistance in tumor cells and some human parasites and the maturation of tumors. From the early 1980s, this gene amplification was used in the industrial production of therapeutic proteins using Chinese hamster ovary (CHO) cells. The most pressing problem in such protein production in mammalian cells is the low productivity of the recombinant protein. To increase productivity, gene amplification cell engineering techniques for the transfected gene are extensively applied, whereby the amplifiable gene is used as a selectable marker for the transfected vector. Firstly the vector, which contains the cDNA of the objective protein and amplifiable marker gene, is introduced into the host cell line. When the selectable and amplifiable gene is amplified, genetically linked sequences containing the objective cDNA are co-amplified. Therefore, a high level of expression of the transfected DNA can be easily attained. In this study, we analyzed the gene amplification mechanism by construction of CHO bacterial artificial chromosome (BAC) library. We determined the structure of gene-amplified location and estimated the gene amplification mechanism.