Identification of a novel temperature sensitive promoter in CHO cells.

BACKGROUND: The Chinese hamster ovary (CHO) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature. RESULTS: Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin) and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp) comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence. CONCLUSIONS: This novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter) is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.

Identification of CHO endogenous promoter elements based on a genomic library approach.

Today the standard technology for the production of many biopharmaceutical products from mammalian cell systems is frequently based on expression vectors that utilize strong mammalian active viral promoters like CMV or SV40 for driving recombinant gene transcription. On one hand these promoters allow very high expression rates, but on the other hand, they can lead to constitutive over-expression of the gene of interest resulting in a permanent stress on the cell. Another drawback is that they are cell cycle-dependent and can be subject to gene silencing which leads to a heterogeneity within the cell population. Here we aim to identify endogenous gene regulatory elements that are capable of controlling the transcription of the foreign gene. For this purpose, a genomic CHO library containing fragments of various lengths was constructed using a shotgun cloning strategy. After enzymatic fragmentation of genomic DNA, fragments encoding potential promoter regions were inserted into a promoterless vector upstream of the neomycin resistance gene. This random pool of library plasmids was transfected back into CHO cells and via cell sorting directly into 96-well plates and G418 (neomycin) selection, positive clones were isolated. A nested PCR of resistant CHO clones resulted in potential fragments which were sequenced. Putative promoter activity was predicted for these sequences by in silico methods and will be proved by re-transfection of reporter constructs into CHO cells.

Single bolus injection of bilirubin improves the clinical outcome in a mouse model of endotoxemia.

Increasing serum levels of biliverdin and bilirubin was shown to be beneficial in settings of inflammation. Bilirubin was shown to be protective in LPS-induced lung injury in rats; however, the exact mechanism remains elusive. Here, we investigated whether a single bolus injection of bilirubin would exert anti-inflammatory effects in a mouse model of endotoxemia. Mice were challenged with sublethal doses (2 mg/kg body weight) of LPS, and the effects of intravenously administered bilirubin (40 mg/kg body weight) were assessed. In contrast to control animals, bilirubin-treated animals fully recovered from endotoxin shock within 24 h. Bilirubin treatment improved the clinical score significantly at all time points assessed, attenuated weight loss, and improved LPS-induced anorexia. Furthermore, bilirubin treatment inhibited LPS-induced leukocyte-endothelial interactions and leukocyte accumulation in various tissues. Expression of inflammatory genes, including endothelial adhesion molecules, but also IL-1beta and TNF-alpha, was significantly reduced in bilirubin-treated animals. Moreover, bilirubin inhibited LPS-induced expression of inflammatory genes in isolated cultured aortic endothelial cells and in bone marrow-derived macrophages. These data show that single-dose administration of bilirubin attenuates tissue injury induced by endotoxin, and that bilirubin, in addition to its antioxidant effects, also exerts potent anti-inflammatory activity.

Artificially designed promoters: understanding the role of spatial features and canonical binding sites in transcription.

The promoter is a key element in gene transcription and regulation. We previously reported that artificial sequences rich in the dinucleotide CpG are sufficient to drive expression in vitro in mammalian cell lines, without requiring canonical binding sites for transcription factor proteins. Here, we report that introducing a promoter organization that alternates in CpGs and regions rich in A and T further increases expression strength, as well as how insertion of specific binding sites makes such sequences respond to induced levels of the transcription factor NFκB. Our findings further contribute to the mechanistic understanding of promoters, as well as how these sequences might be shaped by evolutionary pressure in living organisms.

Exploiting nucleotide composition to engineer promoters.

The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a "proof-of-concept" for custom-designing promoters that are suitable for biotechnological and medical applications.

Structure and basal transcription complex of RNA polymerase II core promoters in the mammalian genome: an overview.

The mammalian core promoter is a sophisticated and crucial component for the regulation of transcription mediated by the RNA polymerase II. It is generally defined as the minimal region of contiguous DNA sequence that is sufficient to accurately initiate a basal level of gene expression. The core promoter represents the ultimate target for nucleation of a functional pre-initiation complex composed of the RNA polymerase II and associated general transcription factors. Among the more than 40 distinct proteins assembling the basal transcription complex, TFIID plays a central role in recognizing and binding specific core promoter elements to support creating an environment that facilitates transcription initiation. Several common DNA motifs, like the TATA box, initiator region, or the downstream promoter element, are found in a subset of core promoters present in various combinations. Another class of promoters that is usually absent of a TATA box is constituted by the so-called CpG islands, which are associated with the majority of protein-coding genes within the mammalian genome.

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.

Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells.

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.