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Gewählte Publikation:

Pontiller, J; Maccani, A; Baumann, M; Klancnik, I; Ernst, W; .
(2010): Identification of CHO endogenous gene regulatory elements.
Mol Biotechnol. 2010; 45(3):235-240 FullText FullText_BOKU

Abstract:
The objective of this approach was to identify new CHO endogenous gene regulatory elements that are capable of regulating foreign gene expression in recombinant CHO host cells. The standard technology for the production of many biopharmaceutical products is frequently based on expression vectors that utilize strong mammalian viral promoters like SV40 or CMV which allow for very high expression rates but this may lead to constitutive over-expression resulting in a permanent stress for the cell. In addition, some heterologous promoters are cell-cycle dependent and can be subject to gene silencing generating heterogeneity within the cell population. Here, we describe the construction of a genomic CHO library and the subsequent identification and isolation of selected target sequences that are believed to be responsible for high level expression of the associated genes. The method that was used to isolate these regions of interest relies on gene specific amplification with primer pairs binding on different genes and the vector sequence. Flanking regions of these fragments were identified through Inverse PCR from fragmented and self-ligated genomic DNA. Expression levels of both the initially derived and the mapped fragments were determined through a luciferase reporter assay.
Autor*innen der BOKU Wien:
Baumann Martina
Ernst Wolfgang
Maccani Andreas
BOKU Gendermonitor:

Find related publications in this database (using NML MeSH Indexing)
Animals;CHO Cells/physiology*;Cloning, Molecular/methods*;Cricetinae;Cricetulus;Electrophoresis, Agar Gel;Gene Library;Genes, Reporter;Polymerase Chain Reaction;Recombinant Proteins/biosynthesis*;Recombinant Proteins/genetics;Regulatory Elements, Transcriptional*;

Find related publications in this database (Keywords)
Endogenous promoter
Chinese hamster ovary cells
Recombinant protein expression
Library construction
DNA fragmentation


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