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Boosting native promoter activities with non-adjacent response-element multimers

Paper ID Volume ID Publish Year Pages File Format Full-Text
24011 43489 2010 9 PDF Available
Title
Boosting native promoter activities with non-adjacent response-element multimers
Abstract

Effective gene therapy requires regulated gene expression and vector safety. We developed a strategy to exponentially increase native promoter activity while retaining inherent regulation by inserting multi-copy response elements (REs) into non-adjacent locations. For the hepatocyte nuclear factor (HNF) 4α-dependent Hnf1a (MODY3) gene, HNF4α stimulation increased from 5- to 90-fold by inserting 3 additional HNF4α REs (H4REs). Constructing a promoter with two 4xH4REs 0.25 kb apart by duplicating the 4xH4RE fragment increased stimulation to >1000-fold. HNF4α-induced protein expression by the duplicate 4xH4RE Hnf1a promoter was comparable to a viral promoter. Converting the two Apolipoprotein C3 (ApoC3) H4REs spaced 0.61 kb apart to 4xH4REs achieved a similar result. Increasing spacing to 2.1 kb with non-promoter DNA abolished the augmentation. Finally, converting the HNF1α RE of the HNF4A (MODY1) P2 promoter to 4xH1RE and adding a second 4xH1RE 0.84 kb upstream increased HNF1α stimulation from 26- to >200-fold. Deleting intervening DNA to produce 0.23-kb spacing increased stimulation to >500-fold. Spaced multi-copy RE motifs is a novel strategy for engineering promoters that boosts activity far beyond other techniques. Augmentation of three promoters suggests that this approach is potentially applicable to other promoters for gene therapy and might obviate the need for viral promoters.

Keywords
bp, base pair; HNF, hepatocyte nuclear factor; H1RE, HNF1 response element; H4RE, HNF4αresponse element; MODY, maturity onset diabetes of the young; RE, response elementPromoter; Gene expression; Multiple response elements; Augmentation strategy; Gene the
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 150, Issue 2, 15 October 2010, Pages 259–267
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