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Identification of a novel endogenous regulatory element in Chinese hamster ovary cells by promoter trap

Paper ID Volume ID Publish Year Pages File Format Full-Text
23498 43444 2013 7 PDF Available
Title
Identification of a novel endogenous regulatory element in Chinese hamster ovary cells by promoter trap
Abstract

•A promoter trap was used to identify endogenously active promoters in CHO cells.•The promoter trap was a promoter-less vector bearing a GFP-HygR cassette.•One fragment isolated was found to drive gene expression in two reporter systems.•A 156-bp element key to promoter activity was 66% as active as the SV-40 promoter.

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.

Keywords
Promoter trap; Endogenous promoter; Chinese hamster ovary cells; Expression system; Biopharmaceutical
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Identification of a novel endogenous regulatory element in Chinese hamster ovary cells by promoter trap
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 167, Issue 3, 10 September 2013, Pages 255–261
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us