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Articles de revues sur le sujet « Transient and stable expression »

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Auteur : Grafiati

Publié le 11 mars 2023

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1

Chen, Ridong, Soon Seog Jeong, Jun Luo et Maria Borovilos. « 20 Transient and Stable Expression of Authentic Human Proteins ». Cytokine 39, n^o 1 (juillet 2007) : 6. http://dx.doi.org/10.1016/j.cyto.2007.07.025.

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Peffley, E. B., M. Hart, Z. Xiang, Z. Hiang, A. El-Sharif, N. Dong et G. C. Phillips. « TRANSIENT EXPRESSION OF GUS IN ONION TRANSFORMANTS ». HortScience 31, n^o 5 (septembre 1996) : 759b—759. http://dx.doi.org/10.21273/hortsci.31.5.759b.

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Particle bombardment using the Bio-Rad PDS-1000/HE system is being investigated as a means to develop a stable transformation system for the bulb onion. Donor materials for bombardments included sterile meristems and radicals from newly germinated seeds, callus of Allium cepa `TG1015', `Sunlite', and `Buffalo', A. fistulosum `Heshiko', and suspension-cultured cell lines with confirmed regeneration capability, as well as regenerated plants of an F1 interspecific hybrid. Transient expression assays using the B-glucuronidase (GUS) reporter gene system were used to optimize the conditions for transformation. Various promoters combined with the GUS coding sequence were tested. Results indicate genotype specificity for promoter expression. Some tissue continued to exhibit GUS activity after several months in culture, indicating potential for achieving stable transformation of onion.

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Chen, Jun, Marc R. Lake, Reza S. Sabet, Wende Niforatos, Steve D. Pratt, Steven C. Cassar, Jing Xu et al. « Utility of Large-Scale Transiently Transfected Cells for Cell-Based High-Throughput Screens to Identify Transient Receptor Potential Channel A1 (TRPA1) Antagonists ». Journal of Biomolecular Screening 12, n^o 1 (12 novembre 2006) : 61–69. http://dx.doi.org/10.1177/1087057106295220.

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Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

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Ju, Xiaoli, Meijia Ren, Keping Chen et Qiang Wang. « Overexpression of c-Myc enhances recombinant protein production in High Five cells after baculovirus infection ». Zeitschrift für Naturforschung C 73, n^o 3-4 (23 février 2018) : 147–51. http://dx.doi.org/10.1515/znc-2017-0076.

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AbstractDue to their numerous advantages, baculovirus expression vector systems (BEVS) have been widely used to express recombinant proteins for different purposes. Different strategies have been adopted to increase recombinant protein production. In this study, we transiently or stably expressed mousec-Mycin High Five cells using a commercial pIB/V5 vector. Under the control of theOpIE2promoter, this vector could enhance recombinant protein production. We found that transient expression ofc-Mycin High Five cells improved recombinant protein production. Furthermore, we established two stable cell lines, High Five-c-Myc #1 and High Five-c-Myc #2, that stably expressed mousec-Myc. We further found that the expression level of the recombinant protein was increased in these stable cell lines compared to control cell lines. These data indicate that overexpressingc-Mycin cells is a promising way to improve recombinant protein production in BEVS.

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Liew, Chee-Gee, Jonathan S. Draper, James Walsh, Harry Moore et Peter W. Andrews. « Transient and Stable Transgene Expression in Human Embryonic Stem Cells ». Stem Cells 25, n^o 6 (juin 2007) : 1521–28. http://dx.doi.org/10.1634/stemcells.2006-0634.

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Liew, Chee-Gee, Jonathan S. Draper, James Walsh, Harry Moore et Peter W. Andrews. « Transient and Stable Transgene Expression in Human Embryonic Stem Cells ». Stem Cells 26, n^o 4 (avril 2008) : 1094. http://dx.doi.org/10.1634/stemcells.2006-0634erratum.

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Mortimer, Cara L., Benjamin Dugdale et James L. Dale. « Updates in inducible transgene expression using viral vectors : from transient to stable expression ». Current Opinion in Biotechnology 32 (avril 2015) : 85–92. http://dx.doi.org/10.1016/j.copbio.2014.11.009.

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Stelzer, Christoph, et Yaakov Benenson. « Precise determination of input-output mapping for multimodal gene circuits using data from transient transfection ». PLOS Computational Biology 16, n^o 11 (30 novembre 2020) : e1008389. http://dx.doi.org/10.1371/journal.pcbi.1008389.

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The mapping of molecular inputs to their molecular outputs (input/output, I/O mapping) is an important characteristic of gene circuits, both natural and synthetic. Experimental determination of such mappings for synthetic circuits is best performed using stably integrated genetic constructs. In mammalian cells, stable integration of complex circuits is a time-consuming process that hampers rapid characterization of multiple circuit variants. On the other hand, transient transfection is quick. However, it is an extremely noisy process and it is unclear whether the obtained data have any relevance to the input/output mapping of a circuit obtained in the case of a stable integration. Here we describe a data processing workflow, Peakfinder algorithm for flow cytometry data (PFAFF), that allows extracting precise input/output mapping from single-cell protein expression data gathered by flow cytometry after a transient transfection. The workflow builds on the numerically-proven observation that the multivariate modes of input and output expression of multi-channel flow cytometry datasets, pre-binned by the expression level of an independent transfection reporter gene, harbor cells with circuit gene copy numbers distributions that depend deterministically on the properties of a bin. We validate our method by simulating flow cytometry data for seven multi-node circuit architectures, including a complex bi-modal circuit, under stable integration and transient transfection scenarios. The workflow applied to the simulated transient transfection data results in similar conclusions to those reached with simulated stable integration data. This indicates that the input/output mapping derived from transient transfection data using our method is an excellent approximation of the ground truth. Thus, the method allows to determine input/output mapping of complex gene network using noisy transient transfection data.

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Bailly, A., G. Spath, V. Bender et M. C. Weiss. « Phenotypic effects of the forced expression of HNF4 and HNF1alpha are conditioned by properties of the recipient cell ». Journal of Cell Science 111, n^o 16 (15 août 1998) : 2411–21. http://dx.doi.org/10.1242/jcs.111.16.2411.

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Tagged versions of HNF4 or HNF1alpha cDNAs in expression vectors have been introduced by transient and stable transfection into three cell lines of hepatic origin that all fail to express these two liver-enriched transcription factors and hepatic functions. C2 and H5 cells are dedifferentiated rat hepatoma variants and WIF12-E cells are human fibroblast-rat hepatoma hybrids with a reduced complement of human chromosomes. Transfectants were analyzed for the expression state of the endogenous genes coding for these transcription factors and for hepatic functions. Each cell line showed a different response to the forced expression of the transcription factors. In C2 cells, no measurable effect was observed, either upon transitory or stable expression. H5 cells reexpressed the endogenous HNF4 gene only upon transient HNF1alpha transfection, and the endogenous HNF1alpha gene only in stable HNF4 transfectants. WIF12-E cells responded to the forced transient or stable expression of either HNF1alpha or HNF4 by cross-activation of the corresponding endogenous gene. In addition, the stable transfectants reexpress HNF3alpha and C/EBPalpha, as well as all of the hepatic functions examined. Hybrid cells similar to WIF12-E had previously been observed to show pleiotropic reexpression of the hepatic phenotype in parallel with loss of human chromosome 2. For the stable WIF12-E transfectants, it was verified that reexpression of the hepatic phenotype was not due to loss of human chromosome 2. The demonstration of reciprocal cross-regulation between HNF4 and HNF1alpha in transient as well as stable transfectants implies that direct effects are involved.

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Alexander, L., H. Lee, M. Rosenzweig, J. U. Jung et R. C. Desrosiers. « EGFP-Containing Vector System that Facilitates Stable and Transient Expression Assays ». BioTechniques 23, n^o 1 (juillet 1997) : 64–66. http://dx.doi.org/10.2144/97231bm12.

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Deroles, SC, et J. Javellana. « Factors influencing transient and stable gene expression inSandersonia aurantiacaHook callus tissue ». New Zealand Journal of Crop and Horticultural Science 39, n^o 1 (mars 2011) : 1–6. http://dx.doi.org/10.1080/01140671.2010.508819.

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BURLAND, T., J. BAILEY, W. DOVE, M. MUKHOPADHYAY et D. PALLOTTA. « Methods for transient and stable expression of heterologous genes in Physarum ». Cell Biology International Reports 16, n^o 11 (1992) : 1111–17. http://dx.doi.org/10.1016/s0309-1651(05)80037-2.

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Hema, M. V., Theertha Prasad et Akella Vani. « Transient expression and stable integration of chimericGusgene in watermelon following electroporation ». Journal of Horticultural Science and Biotechnology 79, n^o 3 (janvier 2004) : 364–69. http://dx.doi.org/10.1080/14620316.2004.11511774.

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14

Duch, Mogens, Kirsten Paludan, Lene Pedersen, Poul Jørgensen, Niels Ole Kjeldgaard et Finn Skou Pedersen. « Determination of transient or stable neo expression levels in mammalian cells ». Gene 95, n^o 2 (novembre 1990) : 285–88. http://dx.doi.org/10.1016/0378-1119(90)90373-y.

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Fibi, MR, P. Hermentin, JU Pauly, L. Lauffer et G. Zettlmeissl. « N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells ». Blood 85, n^o 5 (1 mars 1995) : 1229–36. http://dx.doi.org/10.1182/blood.v85.5.1229.bloodjournal8551229.

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Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.

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16

Franks, T., et RG Birch. « Gene Transfer Into Intact Sugarcane Cells Using Microprojectile Bombardment ». Functional Plant Biology 18, n^o 5 (1991) : 471. http://dx.doi.org/10.1071/pp9910471.

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A microprojectile accelerator has been constructed and used to bombard cultured sugarcane tissues with GUS reporter gene constructs. Design features useful to minimise target tissue damage and variation between shots are described. Transient expression of GUS occurred in pEmuGN-bombarded cells of nonregenerable suspension culture as well as in regenerable embryogenic callus of commercial sugarcane cultivar 463, and in suspension cultures capable of regeneration to plants. Parameters yielding transient GUS expression in up to 1055 cells per bombardment in homogeneous suspension cultures of sugarcane have been established with a mean of 206 expressing cells per bombardment over a series of 8 independent experiments. Approximately 4% of these transiently expressing cells continued to express GUS for extended periods, indicating probable stable transformation of intact cells of the commercial sugarcane cultivar. Microprojectile bombardment appears the most promising of the available gene transfer techniques for practical genetic transformation of sugarcane because most commercial cultivars readily form regenerable callus suitable for bombardment.

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17

Kalos, M., et R. E. Fournier. « Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain. » Molecular and Cellular Biology 15, n^o 1 (janvier 1995) : 198–207. http://dx.doi.org/10.1128/mcb.15.1.198.

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The human apolipoprotein B (apoB) gene resides within a 47.5-kb chromatin domain that is flanked by sequences that bind to the nuclear matrix. These matrix attachment regions (MARs) are boundaries between nuclease-sensitive and -resistant chromatin. As domain boundaries are thought to function as insulator elements, shielding sequences between them from effects of neighboring chromatin, this raised the possibility that the apoB MARs have functions that could be assayed by transfection. To test this possibility, we examined effects of the apoB MARs on transgene expression in transiently and stably transfected rat and human hepatoma cells. The apoB MARs had no effects on expression of transiently transfected reporters, but they altered expression of stably integrated transgenes in dramatic and reproducible ways. Single integrated copies of transgenes that contained the apoB promoter and second intron enhancer, which are sufficient for high-level expression in transient assays, were expressed at low and variable levels in stable transfectant clones. In contrast, transgenes containing the apoB 5' and 3' MARs were expressed at levels nearly 200-fold higher than levels of the minimal reporters in stable transfectants, and expression was position independent. Transgenes that contained the apoB MARs and an additional 3.3 kb of apoB 5' flanking sequence were also expressed in an elevated, position-independent manner. Surprisingly, tandem transgene arrays in multicopy transfectants were transcriptionally inactive. These observations suggest that the apoB MARs function as insulator elements, shielding transgene expression from effects of neighboring chromatin domains.

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Shohag, Md Jahidul Islam, Farhana Zerin Khan, Lin Tang, Yanyan Wei, Zhenli He et Xiaoe Yang. « COVID-19 Crisis : How Can Plant Biotechnology Help ? » Plants 10, n^o 2 (12 février 2021) : 352. http://dx.doi.org/10.3390/plants10020352.

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The emergence of the COVID-19 pandemic has led to significant public health crisis all over the world. The rapid spreading nature and high mortality rate of COVID-19 places a huge pressure on scientists to develop effective diagnostics and therapeutics to control the pandemic. Some scientists working on plant biotechnology together with commercial enterprises for the emergency manufacturing of diagnostics and therapeutics have aimed to fulfill the rapid demand for SARS-CoV-2 protein antigen and antibody through a rapid, scalable technology known as transient/stable expression in plants. Plant biotechnology using transient/stable expression offers a rapid solution to address this crisis through the production of low-cost diagnostics, antiviral drugs, immunotherapy, and vaccines. Transient/stable expression technology for manufacturing plant-based biopharmaceuticals is already established at commercial scale. Here, current opinions regarding how plant biotechnology can help fight against COVID-19 through the production of low-cost diagnostics and therapeutics are discussed.

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19

Naghneh, Ehsan, Es'hagh Pourmaleki et Azam Rahimpour. « Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor-Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells ». Pharmaceutical Sciences 26, n^o 4 (25 décembre 2020) : 393–98. http://dx.doi.org/10.34172/ps.2020.37.

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Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.

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Motohashi, Yu, Ayako Ohashi-Kobayashi, Mayumi Nakanishi-Matsui, Yasuyuki Fujimoto et Masatomo Maeda. « Intracellular Localization of ABC Transporter TAPL Differs between Transient and Stable Expression ». CellBio 03, n^o 02 (2014) : 50–59. http://dx.doi.org/10.4236/cellbio.2014.32006.

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Smith, C. L., T. K. Archer, G. Hamlin-Green et G. L. Hager. « Newly expressed progesterone receptor cannot activate stable, replicated mouse mammary tumor virus templates but acquires transactivation potential upon continuous expression ». Proceedings of the National Academy of Sciences 90, n^o 23 (1 décembre 1993) : 11202–6. http://dx.doi.org/10.1073/pnas.90.23.11202.

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During development and differentiation, the expression of transcription factors is regulated in a temporal fashion. Newly expressed transcription factors must interact productively with target genes organized in chromatin. Although the mechanisms governing factor binding to chromatin templates are not well understood, it is now clear that template access can be dramatically influenced by nucleoprotein structure. We have examined the ability of a well characterized transactivator, the progesterone receptor (PR), to activate the mouse mammary tumor virus (MMTV) promoter organized either in stable, replicating templates that have a highly ordered nucleosome structure or as transiently transfected DNA, which adopts a less-defined structure. If the PR is transiently expressed in cells harboring both replicated and transient MMTV receptor constructs, it cannot significantly activate the stable replicated MMTV template. In contrast, when PR cDNA is stably inserted into the same cells and constitutively expressed, it gains the ability to activate both chromosomal and transiently introduced templates. These results demonstrate that newly expressed PR is not competent to activate the MMTV template in its native nucleoprotein conformation but acquires this ability upon prolonged expression in replicating cells.

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Ward, Alister C., Dora O. McPhee, Melanie M. Condron, Sony Varma, Stephen H. Cody, Sara M. N. Onnebo, Barry H. Paw, Leonard I. Zon et Graham J. Lieschke. « The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish ». Blood 102, n^o 9 (1 novembre 2003) : 3238–40. http://dx.doi.org/10.1182/blood-2003-03-0966.

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AbstractThe spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.

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Malla, Ashwini, Balamurugan Shanmugaraj, Ashutosh Sharma et Sathishkumar Ramalingam. « Production of Genistein in Amaranthus tricolor var. tristis and Spinacia oleracea by Expression of Glycine max Isoflavone Synthase ». Plants 10, n^o 11 (27 octobre 2021) : 2311. http://dx.doi.org/10.3390/plants10112311.

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Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants. It has received considerable attention in recent days due to its health promoting benefits and is known to prevent certain diseases in humans. These isoflavonoids are synthesized from flavonoid intermediates of phenylpropanoid pathway by the enzyme isoflavone synthase. Metabolic engineering of isoflavonoid biosynthesis in non-legume crop plants could offer the health benefits of these compounds in diverse plant species further contributing for crop improvement. The transient expression of heterologous genes in the host is considered as an alternative to stable expression, that can provide a rapid way of studying the pathway engineering for metabolite production and could also act as a production platform for nutraceuticals and biopharmaceuticals. In this study, isoflavone genistein was produced in Amaranthus tricolor var. tristis and Spinacia oleracea by transiently expressing Glycine max isoflavone synthase (GmIFS). The GmIFS gene was cloned in plant expression vector pEarleyGate 102 HA and pEAQ-HT-DEST 3 and transformed into plants by agroinfiltration. The presence of transgene in the agroinfiltrated leaves was confirmed by semiquantitative reverse-transcription polymerase chain reaction. The flavonoid substrate naringenin and isoflavonoid genistein were quantified using high performance liquid chromatography in both wild-type and infiltrated leaf samples of both the plants. The naringenin content varied in the range of 65.5–338.5 nM/g fresh weight, while the accumulation of genistein was observed with varying concentrations from 113 to 182.6 nM/g fresh weight in the agroinfiltrated leaf samples of both A. tricolor var. tristis and S. oleracea. These results indicate that the transient expression of GmIFS gene has led to the synthesis of isoflavonoid genistein in A. tricolor var. tristis and S. oleracea providing an insight that stable expression of this gene could enrich the nutraceutical content in the crop plants. To the best of our knowledge, this is the first report on transient expression of GmIFS gene for the production of genistein in A. tricolor var. tristis and S. oleracea.

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LI, Xiaomei, Vicki VAN PUTTEN, Fariba ZARINETCHI, E. Michael NICKS, Seth THALER, E. Lynn HEASLEY et A. Raphael NEMENOFF. « Suppression of smooth-muscle α-actin expression by platelet-derived growth factor in vascular smooth-muscle cells involves Ras and cytosolic phospholipase A2 ». Biochemical Journal 327, n^o 3 (1 novembre 1997) : 709–16. http://dx.doi.org/10.1042/bj3270709.

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Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle α-actin (SM-α-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-α-actin protein. Arginine vasopressin, which stimulated SM-α-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-α-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-α-actin protein, and stimulation of SM-α-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-α-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-α-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-α-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production.

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Javed, Aadil, Gülseren Özduman, Lokman Varışlı, Bilge Esin Öztürk et Kemal Sami Korkmaz. « HN1 Is Enriched in the S-Phase, Phosphorylated in Mitosis, and Contributes to Cyclin B1 Degradation in Prostate Cancer Cells ». Biology 12, n^o 2 (26 janvier 2023) : 189. http://dx.doi.org/10.3390/biology12020189.

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HN1 has previously been shown as overexpressed in various cancers. In Prostate cancer, it regulates AR signaling and centrosome-related functions. Previously, in two different studies, HN1 expression has been observed as inversely correlated with Cyclin B1. However, HN1 interacting partners and the role of HN1 interactions in cell cycle pathways have not been completely elucidated. Therefore, we used Prostate cancer cell lines again and utilized both transient and stable inducible overexpression systems to delineate the role of HN1 in the cell cycle. HN1 characterization was performed using treatments of kinase inhibitors, western blotting, flow cytometry, immunofluorescence, cellular fractionation, and immunoprecipitation approaches. Our findings suggest that HN1 overexpression before mitosis (post-G2), using both transient and stable expression systems, leads to S-phase accumulation and causes early mitotic exit after post-G2 overexpression. Mechanistically, HN1 interacted with Cyclin B1 and increased its degradation via ubiquitination through stabilized Cdh1, which is a co-factor of the APC/C complex. Stably HN1-expressing cells exhibited a reduced Cdt1 loading onto chromatin, demonstrating an exit from a G1 to S phenotype. We found HN1 and Cdh1 interaction as a new regulator of the Cyclin B1/CDK1 axis in mitotic regulation which can be explored further to dissect the roles of HN1 in the cell cycle.

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Gopal, T. V. « Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures ». Molecular and Cellular Biology 5, n^o 5 (mai 1985) : 1188–90. http://dx.doi.org/10.1128/mcb.5.5.1188-1190.1985.

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A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells.

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Gopal, T. V. « Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures. » Molecular and Cellular Biology 5, n^o 5 (mai 1985) : 1188–90. http://dx.doi.org/10.1128/mcb.5.5.1188.

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A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells.

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Zeng, Jieming, Juan Du, Ying Zhao, Nallasivam Palanisamy et Shu Wang. « Baculoviral Vector-Mediated Transient and Stable Transgene Expression in Human Embryonic Stem Cells ». Stem Cells 25, n^o 4 (avril 2007) : 1055–61. http://dx.doi.org/10.1634/stemcells.2006-0616.

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Joersbo, Morten, et Janne Brunstedt. « Electroporation : Mechanism and transient expression, stable transformation and biological effects in plant protoplasts ». Physiologia Plantarum 81, n^o 2 (février 1991) : 256–64. http://dx.doi.org/10.1111/j.1399-3054.1991.tb02139.x.

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Joersbo, Morten, et Janne Brunstedt. « Electroporation : Mechanism and transient expression, stable transformation and biological effects in plant protoplasts ». Physiologia Plantarum 81, n^o 2 (février 1991) : 256–64. http://dx.doi.org/10.1034/j.1399-3054.1991.810218.x.

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Ponappa, T., A. E. Brzozowski et J. J. Finer. « Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein ». Plant Cell Reports 19, n^o 1 (11 novembre 1999) : 6–12. http://dx.doi.org/10.1007/s002990050702.

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Lackner, Andreas, Kathrin Genta, Herwig Koppensteiner, Irene Herbacek, Klaus Holzmann, Sabine Spiegl-Kreinecker, Walter Berger et Michael Grusch. « A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells ». Analytical Biochemistry 380, n^o 1 (septembre 2008) : 146–48. http://dx.doi.org/10.1016/j.ab.2008.05.020.

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Kopertekh, Lilya, et Joachim Schiemann. « Transient Production of Recombinant Pharmaceutical Proteins in Plants : Evolution and Perspectives ». Current Medicinal Chemistry 26, n^o 3 (26 mars 2019) : 365–80. http://dx.doi.org/10.2174/0929867324666170718114724.

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During the last two decades, the production of pharmaceutical proteins in plants evolved from proof of concept to established technology adopted by several biotechnological companies. This progress is particularly based on intensive research starting stable genetic transformation and moving to transient expression. Due to its advantages in yield and speed of protein production transient expression platforms became the leading plant-based manufacturing technology. Current transient expression methods rely on Agrobacteriummediated delivery of expression vectors into plant cells. In recent years, great advances have been made in the improvement of expression vectors, host cell engineering as well as in the development of commercial manufacturing processes. Several GMP-certified large-scale production facilities exist around the world to utilize agroinfiltration method. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. The great potential of transient expression platform in respect to rapid response to emerging pandemics was demonstrated by the production of experimental ZMapp antibodies against Ebola virus as well as influenza vaccines. This review is focused on current design, status and future perspectives of plant transient expression system for the production of biopharmaceutical proteins.

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Chakrabarty, Romit, Rituparna Banerjee, Sang-Min Chung, Mark Farman, Vitaly Citovsky, Saskia A. Hogenhout, Tzvi Tzfira et Michael Goodin. « pSITE Vectors for Stable Integration or Transient Expression of Autofluorescent Protein Fusions in Plants : Probing Nicotiana benthamiana-Virus Interactions ». Molecular Plant-Microbe Interactions® 20, n^o 7 (juillet 2007) : 740–50. http://dx.doi.org/10.1094/mpmi-20-7-0740.

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Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.

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Kube, D., M. Vockerodt, O. Weber, K. Hell, J. Wolf, B. Haier, F. A. Grässer et al. « Expression of Epstein-Barr Virus Nuclear Antigen 1 Is Associated with Enhanced Expression of CD25 in the Hodgkin Cell Line L428 ». Journal of Virology 73, n^o 2 (1 février 1999) : 1630–36. http://dx.doi.org/10.1128/jvi.73.2.1630-1636.1999.

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ABSTRACT Epstein-Barr virus is associated with several human malignancies including Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s disease (HD). To examine the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1) in the pathogenesis of HD, we transfected the gene into the HD cell line L428. EBNA-1 expression was associated with significantly enhanced CD25 expression (interleukin 2 [IL-2]-receptor α chain) in transient and stably transfected L428 cells but did not affect the expression of IL-2 receptor β and γ chains. There was no up-regulation of the B-cell activation molecules CD23, CD30, CD39, CD40, CD44, CD71, and CD54 (intercellular adhesion molecule 1) or enhanced production of IL-6, IL-10, lymphotoxin alpha, and the soluble form of CD25. Stable EBNA-1-expressing L428 cells were nontumorigenic in SCID mice but showed enhanced lymphoma development in nonobese diabetic-SCID mice compared to mock-transfected cells.

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Maas, Christoph, Jeff Schell et Hans-Henning Steinbiß. « Applications of an optimized monocot expression vector in studying transient gene expression and stable transformation of barley ». Physiologia Plantarum 85, n^o 2 (juin 1992) : 367–73. http://dx.doi.org/10.1111/j.1399-3054.1992.tb04749.x.

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Maas, Christoph, Jeff Schell et Hans-Henning Steinbibeta. « Applications of an optimized monocot expression vector in studying transient gene expression and stable transformation of barley ». Physiologia Plantarum 85, n^o 2 (juin 1992) : 367–73. http://dx.doi.org/10.1034/j.1399-3054.1992.850235.x.

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van der Steege, Gerrit, Maarten Nieboer, Jelto Swaving et M. J. Tempelaar. « Potato granule-bound starch synthase promoter-controlled GUS expression : regulation of expression after transient and stable transformation ». Plant Molecular Biology 20, n^o 1 (octobre 1992) : 19–30. http://dx.doi.org/10.1007/bf00029145.

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Rubiyana, Yana, Retno Damajanti Soejoedono et Adi Santoso. « Enhancement of transient erythropoietin protein expression by valproic acid in CHO‐K1 suspension adapted cells ». Indonesian Journal of Biotechnology 25, n^o 1 (18 juin 2020) : 28. http://dx.doi.org/10.22146/ijbiotech.52621.

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Erythropoietin (EPO) is a therapeutic protein that is widely used to increase red blood cell production in chronic kidney failure. EPO protein can be produced quickly with a transient gene expression system (TGE). However, the titer produced using TGE is usually lower than the stable gene expression system (SGE). It has been known that TGE can be improved by histone deacetylase inhibitors (iHDACs) such as valproic acid (VPA). This study was conducted to examine the VPA effect on EPO protein expression in CHO‐K1 suspension adapted cells and to find the optimum concentration of VPA on transient EPO protein production. EPO proteins was quantified using the enzyme‐linked immunosorbent assay (ELISA) method. The optimization of VPA concentrations showed that VPA increased the EPO protein yield by up to 2‐fold in transient EPO production, and the optimum concentration of VPA was 4 mM. VPA optimization was very helpful to obtain the maximum increase in the transiently expressed protein. Furthermore, this study can be used as a model to produce EPO proteins or other recombinant proteins rapidly with TGE of CHO‐K1 suspension adapted cells.

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Sleeman, Judith E., Paul Ajuh et Angus I. Lamond. « snRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN ». Journal of Cell Science 114, n^o 24 (15 décembre 2001) : 4407–19. http://dx.doi.org/10.1242/jcs.114.24.4407.

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Splicing snRNPs (small nuclear ribonucleoproteins) are essential sub-units of the spliceosome. Here we report the establishment of stable cell lines expressing fluorescently tagged SmB, a core snRNP protein. Analysis of these stable cell lines has allowed us to characterize the nuclear pathway that leads to snRNP accumulation in nuclear speckles and has identified a limiting nucleolar step in the pathway that can be saturated by overexpression of Sm proteins. After nuclear import, newly assembled snRNPs accumulate first in a subset of Cajal bodies that contain both p80-coilin and the survival of motor neurons protein (SMN) and not in bodies that contain p80-coilin but lack SMN. Treatment of cells with leptomycin B (LMB) inhibits both the accumulation of snRNPs in nuclear bodies and their subsequent accumulation in speckles. The formation of Cajal bodies is enhanced by Sm protein expression and the assembly of new snRNPs. Formation of heterokaryons between HeLa cell lines expressing Sm proteins and primary cells that usually lack Cajal bodies results in the detection of Cajal bodies in primary cell nuclei. Transient over-expression of exogenous SmB alone is sufficient to induce correspondingly transient Cajal body formation in primary cells. These data indicate that the level of snRNP protein expression and snRNP assembly, rather than the expression levels of p80-coilin or SMN, may be a key trigger for Cajal body formation.

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Hnatiuk, I. S., O. I. Varchenko, M. F. Parii et Yu V. Symonenko. « Creation of a genetic vector carrying a synthesis bacterial protein gene CAS9 for plant genome editing ». Faktori eksperimental'noi evolucii organizmiv 26 (1 septembre 2020) : 176–82. http://dx.doi.org/10.7124/feeo.v26.1263.

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Aim. To create a genetic construct carrying the bacterial protein Cas9 gene, the reporter β-glucuronidase gus gene, as well as the marker phosphinotricin-N-acetyltransferase bar gene for plant genome editing. Methods. Molecular-biological, biotechnological, microbiological and bioinformatic methods were used in the study; Golden Gate molecular cloning method was used to create genetic constructs. Results. The genetic construct pSPE2053 which carries the Cas9 endonuclease gene, the gus and bar genes was created; the assembly correctness of all vector elements was confirmed by polymerase chain reaction; the construct was transferred to Escherichia coli and Agrobacterium tumefaciens cells; β-glucuronidase gene expression was verified by histochemical analysis after Nicotiana rustica L transient genetic transformation. Conclusions. The created genetic construct can be used to edit the plant genome for both stable and transient genetic transformation to accumulate recombinant Cas9 protein. The guide RNA sequences may be subsequently transferred into such plants using either stable or transient genetic transformation or traditional crossing methods. Keywords: cloning, genetic construction, gus and bar genes, Cas9 endonuclease protein, transient expression.

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Kubomura, K. « Transient Loads Analysis by Dynamic Condensation ». Journal of Applied Mechanics 52, n^o 3 (1 septembre 1985) : 559–64. http://dx.doi.org/10.1115/1.3169101.

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This paper investigates an alternative finite-difference time-integration procedure in structural dynamics. The integral form of the equation of motion, instead of the differential form, is used to develop the finite-difference time-integration method. An expression called dynamic condensation is presented to relate the dynamic response and unknown force at a limited number of degrees of freedom. A stable and accurate method of analysis is derived to determine the transient loads of structural components in a structural system without constructing the system equation of motion.

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Vyacheslavova, A. O., O. N. Mustafaev, A. A. Tyrin, K. R. Shimshilashvili, I. N. Berdichevets, D. M. Shayakhmetova, M. A. Goldenkov, V. S. Fadeev, Yu V. Sheludko et I. V. Goldenkova-Pavlova. « Set of module vectors for stable or transient expression of heterologous genes in plants ». Russian Journal of Genetics 48, n^o 9 (septembre 2012) : 892–901. http://dx.doi.org/10.1134/s1022795412090098.

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Condreay, J. P., S. M. Witherspoon, W. C. Clay et T. A. Kost. « Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector ». Proceedings of the National Academy of Sciences 96, n^o 1 (5 janvier 1999) : 127–32. http://dx.doi.org/10.1073/pnas.96.1.127.

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Norderhaug, Lars, Tove Olafsen, Terje E. Michaelsen et Inger Sandlie. « Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells ». Journal of Immunological Methods 204, n^o 1 (mai 1997) : 77–87. http://dx.doi.org/10.1016/s0022-1759(97)00034-3.

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Sellhorn, George, Zachary Caldwell, Christine Mineart et Leonidas Stamatatos. « Improving the expression of recombinant soluble HIV Envelope glycoproteins using pseudo-stable transient transfection ». Vaccine 28, n^o 2 (décembre 2009) : 430–36. http://dx.doi.org/10.1016/j.vaccine.2009.10.028.

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Tremblay, Reynald, Mary Feng, Rima Menassa, Norman P. A. Huner, Anthony M. Jevnikar et Shengwu Ma. « High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems ». Transgenic Research 20, n^o 2 (18 juin 2010) : 345–56. http://dx.doi.org/10.1007/s11248-010-9419-0.

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Srinivas, L., G. B. Sunil Kumar, T. R. Ganapathi, C. J. Revathi et V. A. Bapat. « Transient and stable expression of hepatitis B surface antigen in tomato (Lycopersicon esculentum L.) ». Plant Biotechnology Reports 2, n^o 1 (6 mars 2008) : 1–6. http://dx.doi.org/10.1007/s11816-008-0041-z.

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Able, Jason A., Carl Rathus et Ian D. Godwin. « The investigation of optimal bombardment parameters for transient and stable transgene expression in Sorghum ». In Vitro Cellular & ; Developmental Biology - Plant 37, n^o 3 (mai 2001) : 341–48. http://dx.doi.org/10.1007/s11627-001-0061-7.

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Mahon, RE, MF Bateson, DA Chamberlain, CM Higgins, RA Drew et JL Dale. « Transformation of an Australian Variety of Carica papaya Using Microprojectile Bombardment ». Functional Plant Biology 23, n^o 6 (1996) : 679. http://dx.doi.org/10.1071/pp9960679.

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We have developed a method for the stable transformation and regeneration of a dioecious Australian cultivar of Carica papaya (papaw or papaya) by microprojectile bombardment. This method was developed after investigation of both zygotic and somatic embryos as target tissue and optimisation of a number of parameters using transient expression of the uidA reporter gene. The tissue culture regime prior to bombardment was critical in optimisation of transformation. Factors such as age of embryos and various treatments prior to bombardment, increased transient expression by up to 22-fold. Highest uidA transient expression results were obtained when somatic embryos 3 weeks since last subculture on solid medium were given a 3 day treatment in liquid medium, and a 2 h osmotic treatment pre- and post-bombardment. Stably transformed plants were obtained 6 months after bombardment using this system. Transformation efficiency was high with two experiments yielding 45% and 37.5% of bombarded plates regenerating plantlets on media containing kanamycin. The presence of both the uidA and aphA genes, (selectable marker) which code for the enzymes β-glucuronidase (GUS), and neomycin phosphotransferase II (NPTII) respectively, was confirmed in regenerated plantlets by Southern hybridisation.

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