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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“对某一基因或某些基因进行选择性、重复的复制，而不增加基因组中其他基因的比例”。基因扩增是真核生物中普遍存在的现象。它在许多生物的发育、肿瘤细胞和某些人类寄生虫耐药的出现以及肿瘤的成熟过程中是一个重要的过程。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)细胞工业化生产治疗性蛋白。在哺乳动物细胞中生产这种蛋白质的最紧迫的问题是重组蛋白质的生产率很低。为了提高生产率，针对转基因的基因扩增细胞工程技术被广泛应用，其中可扩增的基因被用作转基因载体的可选择标记。首先将含有目的蛋白基因和可扩增标记基因的载体导入宿主细胞。当可选择和可扩增的基因被扩增时，包含目的基因的遗传链接序列被共扩增。因此，可以容易地获得高水平的转基因DNA表达。本研究通过构建CHO细菌人工染色体(BAC)文库，对其基因扩增机理进行了分析。确定了基因扩增部位的结构，并对基因扩增机制进行了初步探讨。