Gene Expression in Mammalian Cells and its Applications

Khan, Kishwar Hayat

doi:10.5681/apb.2013.042

Cited by 69 publications

(27 citation statements)

References 54 publications

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“…Human embryonic kidney (HEK293) and rat phaeochromocytoma (PC12) cell lines (American Type Culture Collection (ATCC)), were used because they are capable of post-translational folding and modifications required to express MOP and 5-HT 1A [ 62 , 63 , 64 ]. The experiments were first performed in HEK293 cells, and key findings were validated in PC12 cells.…”

Section: Methodsmentioning

confidence: 99%

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

et al. 2022

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The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: = (80 ± 70) nM), = (200 ± 70) nM, = (100 ± 70) nM and = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain.

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Section: Methodsmentioning

confidence: 99%

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

et al. 2022

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“…Compared with prokaryotic, yeast and insect cells, CHO cells have the following advantages ( Khan 2013 ): 1) CHO cells can grow under the adhere and suspension state with high cell density, which are suitable for large-scale industrial production ( Lai et al, 2013 ): 2) CHO cells are less sensitive to human virus infection ( O'Flaherty et al, 2020 ): 3) The expressed proteins have high similarity with natural proteins in terms of molecular structure, physicochemical properties and biological functions, and the glycosylation is also more similar to that of human-derived cells due to the lack of immunogenic α-galactose epitopes ( Ghaderi et al, 2012 ): 4) CHO cells are fibroblasts with low endogenous protein secretion, which is beneficial to the isolation and purification of recombinant proteins. ( Mohan et al, 2008 ; Li et al, 2018 ).…”

Section: Mammalian Cells and Expression Vectorsmentioning

confidence: 99%

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells

Wei

Mi²,

Jing

et al. 2022

Front. Bioeng. Biotechnol.

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In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.

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“…Among them, expression systems using mammalian cells are the most physiological since expressed protein is expected to undergo post-translational modification, locate to the correct target organelle, and show proper activity. There are several ways to introduce a target gene into mammalian cells, including transfection of recombinant plasmid with calcium phosphate, chemical reagent, or electroporation and use of recombinant adeno- or other viruses ( Khan, 2013 ). These methods have both advantages and disadvantages.…”

Section: Introductionmentioning

confidence: 99%

Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4

et al. 2022

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Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell expression system (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics and pharmacogenomics research.

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Gene Expression in Mammalian Cells and its Applications

Cited by 69 publications

References 54 publications

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells

Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4

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