Annexin-A1 regulates microRNA-26b* and microRNA-562 to directly target NF-κB and angiogenesis in breast cancer cells.

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown.

Inhibition of human cytomegalovirus replication by overexpression of CREB1.

Expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes is regulated by a strong enhancer-containing promoter with multiple binding sites for various transcription factors, including cyclic AMP response element binding protein 1 (CREB1). Here we show that overexpression of CREB1 potently blocked MIE transcription and HCMV replication. Surprisingly, CREB1 still exhibited strong inhibition of the MIE promoter when all five CREB binding sites within the enhancer were mutated, suggesting that CREB1 regulated the MIE gene expression indirectly.

Conserved microRNAs in Chinese hamster ovary cell lines.

MicroRNAs (miRNAs), a class of short (20-24 nt) non-coding RNAs that direct post-transcriptional repression of messenger RNAs, increasingly have been shown to play a key role in regulating cellular physiology. We investigated the prevalence of miRNAs in Chinese hamster ovary (CHO) cells by high-throughput sequencing. Six cDNA libraries of small RNAs from four CHO cell lines were constructed and sequenced by Illumina sequencing.

An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding.

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N-glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-gamma (IFN-gamma).

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production.

Background: The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.

Developing genomic platforms for Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used in recombinant protein production, yet despite their importance in bioprocessing, few genomic resources have been developed for this cell line. Over the past several years, we have made considerable progress in the development of genomic tools for CHO. Using Sanger-based sequencing technology, we have accrued a sequence repertoire of more than 68,000 expressed sequence tags (ESTs), representing more than 28,000 unique CHO transcripts.

Identification and expression analysis of miRNAs during batch culture of HEK-293 cells.

MicroRNAs (miRNAs) are small non-coding RNAs of about 20-24 nucleotides in length. They regulate gene expression negatively and have been implicated in a wide variety of biological processes. To identify potential miRNAs that may influence the growth and proliferation of mammalian cells cultured in bioreactors, we applied miRNA microarray expression profiling technology to batch cultures of HEK293 cells in protein free media.

Quality assessment of cross-species hybridization of CHO transcriptome on a mouse DNA oligo microarray.

DNA microarray technology has been widely utilized for species with extensive genome sequence information available. Given the limited genomic information pertaining to Chinese hamster ovary (CHO) cell line, cross-species hybridization using mouse microarrays provides a viable alternative. In this study, the utility of mouse Affymetrix microarrays for transcriptome profiling in CHO cells was assessed by hybridizing identical sets of cRNA samples from CHO cells on both mouse and CHO Affymetrix microarrays.

Generation of high-level stable transgene expressing human embryonic stem cell lines using Chinese hamster elongation factor-1 alpha promoter system.

The utilization of human embryonic stem cells (hESC) in regenerative medicine largely depends on the development of technologies that will allow efficient genetic manipulation of the cells in vitro. Although a few studies have described the transfection of hESC for generation of reporter lines stably expressing specific transgenes driven by different promoters, the optimal choice of promoter system for driving transgene in hESC has yet to be elucidated. We show for the first time that Chinese hamster elongation factor-1 alpha (CHEF1) promoter robustly drove reporter gene expression higher than the human elongation factor 1 alpha (hEF1 alpha), other constitutive Chinese hamster promoters, human cytomegalovirus (CMV) immediate early enhancer/promoter and SV40 promoters in hESC by quantitative analysis.

Inactivating FruR global regulator in plasmid-bearing Escherichia coli alters metabolic gene expression and improves growth rate.

The introduction of plasmids into Escherichia coli is known to impose a metabolic burden, which diminishes the growth rate. This effect could arise from perturbation of the central metabolic pathways, which supply precursors and energy for macromolecule synthesis. We knocked out a global regulator of central metabolism, FruR (also called Cra), to assess its phenotypic effect in E.

Comparative transcriptional analysis of mouse hybridoma and recombinant Chinese hamster ovary cells undergoing butyrate treatment.

DNA microarray based transcriptome analysis has become widely used in biomedical research; however, the lack of DNA sequence information available for Chinese hamster ovary (CHO) cells has hampered the application of microarrays for this cell line widely used for recombinant therapeutic protein production. We have constructed an expressed sequence tag (EST) based CHO DNA microarray and employed it for comparative transcriptome analysis of CHO cells and mouse hybridoma cells treated with sodium butyrate. Cross-species hybridization of CHO transcripts to mouse DNA microarrays was also performed to assess the utility of cross-species microarray.

Transcriptome and proteome profiling to understanding the biology of high productivity CHO cells.

A combined transcriptome and proteome analysis was carried out to identify key genes and proteins differentially expressed in Chinese hamster ovary (CHO) cells producing high and low levels of dhfr-GFP fusion protein. Comparison of transcript levels was performed using a proprietary 15K CHO cDNA microarray chip, whereas proteomic analysis was performed using iTRAQ quantitative protein profiling technique. Microarray analysis revealed 77 differentially expressed genes, with 53 genes upregulated and 24 genes downregulated.

Transcriptional profiling of batch and fed-batch protein-free 293-HEK cultures.

Dynamic nutrient feeding to control glutamine at low levels in protein-free fed-batch cultures of 293-human embryonic kidney (HEK) cells achieved cell concentrations of 6 x 10(6) cells/ml. This represented a 4-fold improvement in cell concentration compared to batch cultures. Reduction in glutamine and glucose consumption, as well as lactate and ammonia production, were also observed in these fed-batch cultures.

Specific detection of residual CHO host cell DNA by real-time PCR.

Chinese hamster ovary cells have been widely used to manufacture recombinant proteins for human therapeutic use. A sensitive quantitative real-time polymerase chain reaction assay for the detection of residual Chinese hamster (Cricetulus griseus) DNA is presented in this paper. The assay is reasonably affordable and can be adapted for high-throughput screening using 96-well format.

Targeting early apoptotic genes in batch and fed-batch CHO cell cultures.

Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, were identified and CHO GT (Gene Targeted) cell lines were developed by targeting these four genes. Two were CHO GT(O) cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GT(KD) cell lines involving knockdown of Alg-2 and Requiem which are pro-apoptotic genes using small interfering RNA (siRNA) technology. Comparisons of these CHO GT cell lines with the parental cell line in batch culture (BC) and fed-batch culture (FBC) were performed.

Glutamine or glucose starvation in hybridoma cultures induces death receptor and mitochondrial apoptotic pathways.

Glutamine and glucose are often controlled at low levels in fed-batch strategies to limit ammonia and lactate accumulation and improve productivity of mammalian cell cultures. However, this risks triggering apoptosis if cells are depleted of glutamine or glucose. To examine the apoptosis cascade during glutamine or glucose limitation, the transcriptional profile of FAS, FASL, FADD, FLIP, BAX, p53 and PEG3 in CRL 1606 hybridoma culture was investigated using quantitative real-time PCR.

Hcc-2, a novel mammalian ER thioredoxin that is differentially expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. Thus there is great interest to identify novel HCC diagnostic markers for early detection of the disease and tumour specific associated proteins as potential therapeutic targets in the treatment of HCC. Currently, we are screening for early biomarkers as well as studying the development of HCC by identifying the differentially expressed proteins of HCC tissues during different stages of disease progression.

A novel normalization method for effective removal of systematic variation in microarray data.

Normalization of cDNA and oligonucleotide microarray data has become a standard procedure to offset non-biological differences between two samples for accurate identification of differentially expressed genes. Although there are many normalization techniques available, their ability to accurately remove systematic variation has not been sufficiently evaluated. In this study, we performed experimental validation of various normalization methods in order to assess their ability to accurately offset non-biological differences (systematic variation).

Zinc as an insulin replacement in hybridoma cultures.

There are many advantages to the use of protein-free media for biologics production, including a reduced risk of viral contamination from animal-derived proteins and simplification of downstream purification. In the course of developing protein-free media for hybridoma and myeloma cells, zinc was found to be an effective replacement for insulin, with no negative impact on viable cell density and antibody production. Transcript profiling using DNA microarrays indicated no major change in the global expression profile between the insulin and zinc-supplemented cultures, which is consistent with their similar growth and metabolic characteristics.

Elevation of gamma-glutamyltransferase activity in 293 HEK cells constitutively expressing antisense glutaminase mRNA.

Previous studies have shown that the use of dynamic nutrient feeding to maintain glutamine at low levels in fed-batch cultures reduced the overflow of glutamine metabolism. This strategy resulted in the shift of metabolism towards an energetically more efficient state signified by reduced lactate and ammonia production and thus achieving a higher cell density for enhanced productivity. In an effort to mimic the metabolic changes effected by this fed-batch strategy at the molecular level, 293 HEK cells were engineered via stable transfection with an antisense fragment of the rat phosphate-dependent glutaminase (PDG) gene.

EST sequencing for gene discovery in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are one of the most important cell lines in biological research, and are the most widely used host for industrial production of recombinant therapeutic proteins. Despite their extensive applications, little sequence information is available for molecular based research. To facilitate gene discovery and genetic engineering, two cDNA libraries were constructed from three CHO cell lines grown under various conditions.