title: Identification of a novel endogenous regulatory element in Chinese hamster ovary cells by promoter trap.
abstract: The objective of this study was to identify and isolate endogenous promoters in Chinese hamster ovary (CHO) cells using a promoter trap approach. A promoter-less vector harboring a green fluorescent protein (GFP)-hygromycin resistance gene cassette was designed and transfected into CHO cells. Putative promoters were identified by selecting for GFP(+) clones under hygromycin selection. Genomic DNA from these clones was then digested and self-ligated to give rise to a plasmid carrying the putative promoter sequence as well as elements for replication in E. coli. Functional promoter sequences were subsequently identified by screening the recovered plasmids for their ability to drive GFP expression upon re-transfection into CHO cells. One of the fragments isolated through this approach was found to drive gene expression in two different reporter systems. Further dissection of the fragment led to the identification of a 156-bp element that was four-fold more active than the full-length fragment and 66% as active as the SV-40 promoter. Thus, promoter trap represents an effective strategy for identifying endogenous regulatory regions that can potentially be incorporated into expression vectors to augment expression of recombinant biopharmaceuticals.
authors.affiliation: BioProcess Development, Merck & Co., 1011 Morris Avenue, Union, NJ 07083, USA.
authors.forename: Janice
authors.initials: J
authors.lastname: Chen
authors.forename: Jonathon
authors.initials: J
authors.lastname: Haverty
authors.forename: Liang
authors.initials: L
authors.lastname: Deng
authors.forename: Guanghua
authors.initials: G
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Qiu
authors.forename: Zhong
authors.initials: Z
authors.lastname: Liu
authors.forename: Shuangping
authors.initials: S
authors.lastname: Shi
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D050436:Regulatory Elements, Transcriptional; D017422:Sequence Analysis, DNA
chemical_list: D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins
keywords: Biopharmaceutical; Chinese hamster ovary cells; Endogenous promoter; Expression system; Promoter trap