Identification of CHO Endogenous Gene Regulatory Elements

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.

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

“…Previous efforts at identifying endogenous CHO regulatory elements have utilized genomic library approaches, a laborious method that involves shotgun cloning of genomic fragments into a reporter vector (Pontiller et al, 2008(Pontiller et al, , 2010. By comparison, the promoter trap approach presented here is more streamlined and has a major advantage over the genomic library approach in that only transcriptionally active endogenous promoters are selected.…”

“…Individual clones exhibiting reporter activity (such as antibiotic resistance) are recovered, and the original genomic DNA fragments are isolated and sequenced (Pontiller et al, 2008(Pontiller et al, , 2010. However, this approach is very labor-intensive and can yield potentially confounding results.…”

Promoter and Terminator Discovery and Engineering

“…The advent of genome sequencing and annotation (especially of hosts such as Escherichia coli and S. cerevisiae) allowed for the rapid discovery of endogenous promoters, especially when coupled with mRNA quantification methods. In a similar fashion, promoters for more complex systems such as mammalian hosts have largely been discovered via high-throughput screening methods such as "promoter trapping [9][10][11]." This approach typically involves random integration of a promoter-less vector containing GFP followed by fluorescence-based selection to determine adjacent, upstream regions of the genome that enable transcription.…”

Selecting an appropriate method for expressing S locus F-box-S2 recombinant protein

“…Among the available host expression systems, Escherichia coli and cultivated mammalian cells are considered most widely used [24], [25], [26]. Advantages of using a mammalian expression system include its ability for post-translational modifications (PTMs) such as correct protein folding and glycosylation.…”

“…Advantages of using a mammalian expression system include its ability for post-translational modifications (PTMs) such as correct protein folding and glycosylation. The protein yield of mammalian expression systems can be optimized by the use of right cell line, expression vector, promoter elements and transfection efficiency of the mammalian cells [25], [26], [27].…”