Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange.

Site-specific recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines, this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here, we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site.

Profiling highly conserved microrna expression in recombinant IgG-producing and parental Chinese hamster ovary cells.

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY™ miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT-PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44-derived CHO lines producing a recombinant human IgG. We observed that miR-221 and miR-222 were significantly downregulated in all IgG-producing cell lines when compared with parental DG44, whereas miR-125b was significantly downregulated in one IgG-producing line. In another IgG-producing line, miR-19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let-7b and miR-221 were significantly downregulated. In parental CHO K1, let-7b, miR-15b, and miR-17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering.

Using microarray technology to select housekeeping genes in Chinese hamster ovary cells.

In the present study, we have identified species-specific housekeeping genes (HKGs) for Chinese Hamster Ovary (CHO) cells using data from microarray gene expression profiling. HKGs suitable for quantitative RT-PCR normalization should display relatively stable expression levels across experimental conditions. We analyzed transcription profiles of several IgG-producing recombinant CHO cell lines under numerous culture conditions using a custom CHO DNA microarray platform and calculated relative expression variability from 124 microarrays. We selected a novel panel of candidate HKGs based on their low variability in expression from the microarray data. Compared to three traditional HKGs (Gapdh, Actb, and B2m), the majority of genes on this panel demonstrated lower or equal variability. Each candidate HKG was then validated using qRT-PCR. Final selection of CHO-specific HKGs include Actr5, Eif3i, Hirip3, Pabpn1, Vezt, Cog1, and Yaf2. The results reported here provide a useful tool for gene expression analyses in CHO cells, a critical expression platform used in biotherapeutics.

Serine protease inhibitor 6-deficient mice have increased neutrophil immunity to Pseudomonas aeruginosa.

Inflammation is a localized, protective response to trauma or microbial invasion that destroys the injurious agent and the injured tissue. Neutrophil elastase (NE), a serine protease stored in the azurophil granules of polymorphonuclear neutrophils, digests microbes after phagocytosis. NE can also digest microbes extracellularly but is associated with tissue damage and inflammatory disease. In this study, we show that polymorphonuclear neutrophils from mice deficient in serine protease inhibitor 6, a weak intracellular NE inhibitor, had increased susceptibility to self-inflicted lysis because of increased NE activity. The resulting transient increase in local extracellular NE activity was within a narrow range that resulted in the clearance of Pseudomonas aeruginosa but did not damage the lung. Therefore, deficiency in a weak intracellular inhibitor of NE results in an acute inflammatory response that protects from P. aeruginosa but does not cause lung disease.

Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation.

The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.

Protein 4.1B/differentially expressed in adenocarcinoma of the lung-1 functions as a growth suppressor in meningioma cells by activating Rac1-dependent c-Jun-NH(2)-kinase signaling.

Meningiomas are the second most common brain tumor in adults, yet comparatively little is presently known about the dysregulated growth control pathways involved in their formation and progression. One of the most frequently observed genetic changes in benign meningioma involves loss of protein 4.1B expression. Previous studies from our laboratory have shown that protein 4.1B growth suppression in meningioma is associated with the activation of the c-Jun-NH(2)-kinase (JNK) pathway and requires localization of a small unique region (U2 domain) of protein 4.1B to the plasma membrane. To define the relationship between protein 4.1B expression and JNK activation, as well as to determine the mechanism of JNK activation by protein 4.1B, we used a combination of genetic and pharmacologic approaches. In this report, we show that protein 4.1B/differentially expressed in adenocarcinoma of the lung-1 (DAL-1) expression in meningioma cells in vitro results in JNK activation, which requires the sequential activation of Src, Rac1, and JNK. In addition, inhibition of Rac1 or JNK activation abrogates protein 4.1B/DAL-1 growth suppression and cyclin A regulation. Last, protein 4.1B/DAL-1 regulation of this critical growth control pathway in meningioma cells requires the presence of the U2 domain. Collectively, these observations provide the first mechanistic insights into the function of protein 4.1B as a growth regulator in meningioma cells.

Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity.

After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.

Deficiency in neutrophil elastase does not impair neutrophil recruitment to inflamed sites.

To reach the sites of inflammation, neutrophils traverse the endothelium, its underlying basement membrane, and other barriers depending on the localization of the insulting agent. Whether neutrophil elastase (NE) plays a role in neutrophil recruitment to inflamed sites is still debatable. By exploiting mice deficient in NE (NE(-/-)), we sought to address this dilemma. We recruited neutrophils to the lungs or the peritoneum of wild-type (WT) or NE(-/-) mice by intranasal or intraperitoneal challenge with Pseudomonas aeruginosa or its lipopolysaccharide. At designated times post-inoculation (0, 4, 24, and 48 h), groups of mice were killed to assess changes in leukocyte counts and inflammatory responses. NE(-/-) and WT mice had normal circulating leukocyte numbers including neutrophils and changes in the hemograms in the setting of acute inflammation were indistinguishable. Analyses of lung tissues or fluids from the lungs and peritoneum found that regardless of the inflammatory model, the leukocyte counts including neutrophils and the inflammatory response were similar in NE(-/-) and WT mice at all time points. In vitro, neutrophils isolated from the lungs or the peritoneum of NE(-/-) and WT mice had comparable chemotactic and respiratory-burst functions and migrated normally through Matrigel in response to various stimuli. Interestingly, preincubation of human peripheral blood neutrophils with NE physiologic inhibitors did not alter the migration of the cells through Matrigel. In sum, our findings present the first in vivo description that the absence of NE does not impair neutrophil recruitment to inflamed sites and that NE is not required for basement membrane transmigration of neutrophils.