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

基因扩增染色体定位高表达载体的构建及其在人体细胞中的应用

基本信息

批准号：
17560689
负责人：
OMASA Takeshi
金额：
$ 2.39万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17560689/
关键词：
gene amplification BAC library Chinese hamster ovary cell chromosome MTX CHO(Chinese hamster ovary)CHO (Chinese hamster ovary)

项目摘要

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.

基因扩增是指“选择性地、重复地复制某个基因或某些基因，而基因组中其他基因不按比例增加”。基因扩增是真核生物中普遍存在的现象。它是许多生物体发育、肿瘤细胞和某些人类寄生虫耐药性的出现以及肿瘤成熟的重要过程。从 20 世纪 80 年代初开始，这种基因扩增就被用于利用中国仓鼠卵巢 (CHO) 细胞进行治疗性蛋白质的工业化生产。在哺乳动物细胞中生产此类蛋白质的最紧迫问题是重组蛋白的低生产率。为了提高生产率，广泛应用转染基因的基因扩增细胞工程技术，从而将可扩增基因用作转染载体的选择标记。首先，将含有目标蛋白的cDNA和可扩增标记基因的载体引入宿主细胞系。当选择性和可扩增基因被扩增时，包含目标cDNA的遗传连锁序列被共扩增。因此，可以容易地获得转染DNA的高水平表达。本研究通过构建CHO细菌人工染色体（BAC）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机制。