Identification of CHO endogenous gene regulatory elements.
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.
Pontiller
Jens
J
Department of Biotechnology, Austrian Center of Biopharmaceutical Technology, University of Natural Resources and Applied Life Sciences, Vienna, Austria. jens.pontiller@boku.ac.at
Maccani
Andreas
A
Baumann
Martina
M
Klancnik
Ingo
I
Ernst
Wolfgang
W
D000818:Animals; D016466:CHO Cells / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D017930:Genes, Reporter; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D050436:Regulatory Elements, Transcriptional
D011994:Recombinant Proteins