An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells
An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells
复制标题
DOI：
10.1016/s0378-1119(01)00772-7
复制
发表时间：
2001-12-12
期刊：
影响因子：
3.5
通讯作者：
Thammana, P
中科院分区：
文献类型：
--
作者：
Koduri, RK;Miller, JT;Thammana, P
我们利用反向遗传学从重组中国仓鼠卵巢 (CHO) 细胞系中克隆了位于质粒整合位点两侧的 5.0 kb 基因组 DNA 热点 HIRPE（增加重组蛋白表达的热点）。对 5.0 kb 片段的 DNA 序列分析表明，HIRPE 富含重复元件、Alu 样序列和基质相关区域，这些区域已知与许多哺乳动物系统中的转录活性区域连接。描述了同源重组载体 pTV1 的构建，该载体含有 5.0 kb HIRPE 基因组 DNA、重组基因人 CTLA4-Ig 和作为正选择标记的 dhfr 基因。据观察，pTV1 载体将 CTLA4Ig 基因靶向 CHO 基因组中的首选基因座，有助于在转染的 CHO 细胞中实现高重组基因表达。初步研究表明，与亲本细胞系的观察结果类似，分析的 pTV1 生成的转染瘤似乎在质粒整合位点存在基因组 DNA 的反向重复。 (C) 2001 年由 Elsevier Science B.V. 出版
We employed reverse genetics to clone a 5.0 kb genomic DNA hot spot HIRPE (hot spot for increased recombinant protein expression) flanking the plasmid integration site from a recombinant Chinese hamster ovary (CHO) cell line. DNA sequence analysis of the 5.0 kb fragment revealed that HIRPE is enriched for repetitive elements, Alu-like sequences and matrix-associated regions that are known to be linked with transcriptionally active regions in a number of mammalian systems. The construction of a homologous recombination vector, pTV1, containing the 5.0 kb HIRPE genomic DNA, a recombinant gene human CTLA4-Ig, and the dhfr gene as a positive selection marker is described. It was observed that the pTV1 vector targeted the CTLA4Ig gene to a preferred locus in the CHO genome contributing to high recombinant gene expression in transfected CHO cells. Preliminary studies suggest that similar to the observation with the parental cell line, pTV1-generated transfectomas that were analyzed appear to harbor an inverted duplication of the genomic DNA at the plasmid integration site. (C) 2001 Published by Elsevier Science B.V.