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Alternative Promoters Regulate Cold Inducible RNA-Binding (CIRP) Gene Expression and Enhance Transgene Expression in Mammalian Cells

Al-Fageeh, Mohamed B., Smales, Christopher Mark (2013) Alternative Promoters Regulate Cold Inducible RNA-Binding (CIRP) Gene Expression and Enhance Transgene Expression in Mammalian Cells. Molecular Biotechnology, 54 (2). pp. 238-249. ISSN 1073-6085. (doi:10.1007/s12033-013-9649-5) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) (KAR id:33895)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided. (Contact us about this Publication)
Official URL
http://dx.doi.org/10.1007/s12033-013-9649-5

Abstract

The use of a temperature shift cultivation to enhance recombinant protein yield is widely utilised in the bioprocessing industry. The responses of mammalian cells to heat stress are well characterized; however, the equivalent cold stress responses are not. In particular, the transcriptional mechanisms that lead to enhanced gene-specific expression upon cold stress have yet to be elucidated. We report here in silico and experimental identification and characterization of transcriptional control elements that regulate cold inducible RNA-binding (CIRP) gene expression and demonstrate these can be used for enhanced transgene expression. In silico analysis identified the core CIRP promoter and a number of conserved transcription factor-binding sites across mammalian species. The core promoter was confirmed by experimental studies that located the basal transcriptional regulatory elements of CIRP within 264 nucleotides upstream of the transcription start site. Deletion analysis of a fragment from -264 to -64 that contained two putative CAAT-binding sites abolished promoter activity. A second promoter was identified in the region -452 to -264 of the transcription start site which was able to drive transcription independent of the core promoter. As the two CIRP promoters were transcriptionally active and possibly cold responsive, we used electrophoretic mobility shift assays to show that both promoter regions are able to bind factors within a nuclear extract in a dose-dependent manner and that the formation of these complexes was specific to the promoter regions. Finally, we successfully demonstrate using a reporter gene approach that enhanced transgene expression can be achieved using the identified CIRP promoter.

Item Type: Article
DOI/Identification number: 10.1007/s12033-013-9649-5
Uncontrolled keywords: Cold-shock, Cold-inducible RNA-binding protein, Promoters, Transgene expression, Gene expression
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Mark Smales
Date Deposited: 13 May 2013 15:09 UTC
Last Modified: 29 May 2019 10:11 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/33895 (The current URI for this page, for reference purposes)
Smales, Christopher Mark: https://orcid.org/0000-0002-2762-4724
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