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The functional cell surface expression of recombinant odorant receptors typically has been investigated by expressing N-terminally extended, "tagged" receptors in test cell systems, using antibody-based immunocytochemistry or flow cytometry, and by measuring odorant/receptor-induced cAMP signaling, mostly by an odorant/receptor-induced and cAMP signaling-dependent transcriptional activation of a luciferase-based luminescence assay. In the present protocol, we explain a method to measure the cell-surface expression and signaling of recombinant odorant receptors carrying a bi-functional, N-terminal 'IL-6-HaloTag ® '. IL-6, being a secreted cytokine, facilitates functional cell surface expression of recombinant HaloTag ® -odorant receptors, and the HaloTag ® protein serves as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by antibody-independent, chemical live-cell staining and flow cytometry. Here, we describe how to measure the cell surface expression of recombinant IL-6-HaloTag ® -odorant receptors in HEK-293 cells or NxG 108CC15 cells, by live-cell staining and flow cytometry, and how to measure an odorant-induced activation of these receptors by the fast, real-time, luminescence-based GloSensor ® cAMP assay., Competing Interests: Competing interests: The authors have declared that no competing interests exist.

Summary: Background: A confirmatory rabies diagnosis can be achieved by rapid virus isolation in cell culture using brain tissue from the suspect animal. Several cell lines have been used for this purpose and the murine neuroblastoma cell line Neuro-2a has been found to be the most sensitive. The human embryonic kidney cell line HEK-293 is known to express several neuronal proteins and is believed to be of neuronal origin. We hypothesized that this cell line could be susceptible to rabies virus, which is highly neurotropic. Methods: First we tested the sensitivity of HEK-293 cells to the laboratory strain, challenge virus standard (CVS). We then tested 120 brain samples from different animals and humans suspected to have died of rabies by fluorescent antibody test (FAT). Both FAT-positive and FAT-negative brains were tested for virus isolation using Neuro-2a, BHK-21, and HEK-293 cell lines and also by mouse inoculation. Results: There was 100% correlation between FAT, virus isolation in Neuro-2a and HEK-293 cells, and mouse inoculation. However, the rate of virus isolation in the BHK-21 cell line was only 28% when compared to the other cell lines. The sensitivity of HEK-293 to CVS strain of virus was similar to that of Neuro-2a. Conclusions: We conclude that the HEK-293 cell line is as sensitive as the Neuro-2a cell line for the rapid isolation of rabies virus and may serve as an alternative cell line for rabies diagnosis and future research. [ABSTRACT FROM AUTHOR]

The Human Embryonic Kidney 293 cell line (HEK-293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistive monolayers necessary for studying transepithelial ion transport, they are nevertheless valuable in studying individual epithelial ion transporters. We report the use of HEK-293 cells to study the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. While HEK-293 cells endogenously express mRNA for the Cl(-) channels, ClC-2 and TMEM16A, they neither express CFTR mRNA nor protein. Therefore, we stably transfected HEK-293 cells with EGFP-CFTR (HEK-CFTR) and demonstrated CFTR function by measuring forskolin-stimulated iodide efflux. This efflux was inhibited by CFTRinh172, and the protein kinase A inhibitor H89, but not by Ca(2+) chelation. In contrast to intestinal epithelia, forskolin stimulation does not increase surface CFTR expression and does not require intact microtubules in HEK-CFTR. To investigate the role of an endogenous GαS-coupled receptor, we examined the bile acid receptor, TGR5. Although HEK-CFTR cells express TGR5, the potent TGR5 agonist lithocholic acid (LCA; 5-500 μmol/L) did not activate CFTR. Furthermore, forskolin, but not LCA, increased [cAMP]i in HEK-CFTR suggesting that endogenous TGR5 may not be functionally linked to GαS. However, LCA did increase [Ca(2+)]i and interestingly, abolished forskolin-stimulated iodide efflux. Thus, we propose that the stable HEK-CFTR cell line is a useful model to study the multiple signaling pathways that regulate CFTR., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Transient expression systems in mammalian cells have become the method of choice for producing research quantities of antibodies. Both the speed and yield of the available transient systems and the natural posttranslational modifications favor these systems above expression in lower eukaryotes, prokaryotes or stable cell lines. We describe an optimized mammalian transient expression system, capable of producing up to 400mg/L of native secreted antibodies in less than a week. The system is composed of commercially available components and is based on expression in the fast growing suspension cell line, FreeStyle™ 293-F (HEK-293F). The method depends on an optimal combination of a gene transfer method, an expression vector and cotransfection with expression enhancing plasmids, encoding the large T antigen of the SV40 virus and the cell cycle inhibitors p21 and p27. Optimization of all components of the expression system, by experimental design techniques, yielded maximal expression levels (including antibody isotypes IgG1, 2, 3, 4 and Fab fragments of various species). Expression volumes were scalable from 0.1 ml up to 1.2L in a simple shaker flask system in animal component free, low protein medium, enabling consistent production of relatively high amounts of a large number of native antibodies., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The interleukin 1 receptor-associated kinase 4 (IRAK4) is an essential factor for TLR-mediated activation of the host's immune functions subsequent to pathogen contact. We have characterized the respective cDNA and gene sequences from three salmonid species, salmon, rainbow trout and maraena whitefish. The gene from salmon is structured into eleven exons, as is the mammalian homologue, while exons have been fused in the genes from the two other salmonid species. Rainbow trout expresses also a pseudogene at low levels. Its basic structure resembles more closely the primordial gene than the functional copy does. The N-terminal death domain and the C-terminal protein kinase domain of the factors are better conserved throughout evolution than the linker domain. The deduced amino acid sequences of the factors from all three species group together in an evolutionary tree of IRAK4 factors. Scrutinizing expression and function of IRAK4 from rainbow trout, we found its highest expression in head kidney and spleen and lowest expression in muscle tissue. Infecting fish with Aeromonas salmonicida did not modulate its expression during 72 h of observation. Expression of a GFP-tagged trout IRAK4 revealed, expectedly, its cytoplasmic localization in human HEK-293 cells. However, this factor significantly quenched in a dose-dependent fashion not only the pathogen-induced stimulation of NF-κB factors in the HEK-293 reconstitution system of TLR2 signaling, but also the basal NF-κB levels in unstimulated control cells. Our data unexpectedly imply that IRAK4 is involved in establishing threshold levels of active NF-κB in resting cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The nephrotoxicity limits the clinical application of cisplatin. Human organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins (MATEs) work in concert in the elimination of cationic drugs such as cisplatin from the kidney. We hypothesized that co-administration of ondansetron would have an effect on cisplatin nephrotoxicity by altering the function of cisplatin transporters. The inhibitory potencies of ondansetron on metformin accumulation mediated by OCT2 and MATEs were determined in the stable HEK-293 cells expressing these transporters. The effects of ondansetron on drug disposition in vivo were examined by conducting the pharmacokinetics of metformin, a classical substrate for OCTs and MATEs, in wild-type and Mate1-/- mice. The nephrotoxicity was assessed in the wild-type and Mate1-/- mice received cisplatin with and without ondansetron. Both MATEs, including human MATE1, human MATE2-K, and mouse Mate1, and OCT2 (human and mouse) were subject to ondansetron inhibition, with much greater potencies by ondansetron on MATEs. Ondansetron significantly increased tissue accumulation and pharmacokinetic exposure of metformin in wild-type but not in Mate1-/- mice. Moreover, ondansetron treatment significantly enhanced renal accumulation of cisplatin and cisplatin-induced nephrotoxicity which were indicated by increased levels of biochemical and molecular biomarkers and more severe pathohistological changes in mice. Similar increases in nephrotoxicity were caused by genetic deficiency of MATE function in mice. Therefore, the potent inhibition of MATEs by ondansetron enhances the nephrotoxicity associated with cisplatin treatment in mice. Potential nephrotoxic effects of combining the chemotherapeutic cisplatin and the antiemetic 5-hydroxytryptamine-3 (5-HT3) receptor antagonists, such as ondansetron, should be investigated in patients., (© 2013.)

Cystathionine β-synthase (CBS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by a deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)) found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype., (© 2013 Elsevier B.V. All rights reserved.)

Background: The role of organic anion transporting polypeptide 5A1 (OATP5A1) a member of a family of drug transporters that mediate cellular uptake of drugs has not been characterized so far., Methods: Gene expression levels of OATP5A1 in small cell lung cancer (SCLC) cell lines were determined by real-time qPCR and chemosensitivity of HEK-293-SLCO5A1-transfected cells to satraplatin in MTT assays., Results: Significant expression of this transporter was found at the mRNA level, primarily in drug-resistant SCLC cells, and SLCO5A1-transfected HEK-293 cells showed higher resistance to satraplatin. OATP5A1 is found preferentially in cytoplasmic membranes of tumor cells, including SCLC., Conclusions: OATP5A1 seems to effect intracellular transport of drugs and may participate in chemoresistance of SCLC by sequestration, rather than mediating cellular uptake. Since satraplatin failed to improve survival in SCLC patients, the relation of OATP5A1 expression to clinical drug resistance and its use as marker of chemoresistance should be further investigated.