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Eisenhut P, Marx N, Borsi G, Papež M, Ruggeri C, Baumann M, Borth N. Corrigendum to "Manipulating gene expression levels in mammalian cell factories: An outline of synthetic molecular toolboxes to achieve multiplexed control" [New Biotechnol 79 (2024) 1-19]. N Biotechnol 2024; 84:30-36. [PMID: 39332183 DOI: 10.1016/j.nbt.2024.09.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/29/2024]
Affiliation(s)
- Peter Eisenhut
- Austrian Centre of Industrial Biotechnology (acib GmbH), Muthgasse 11, 1190 Vienna, Austria
| | - Nicolas Marx
- BOKU University of Natural Resources and Life Sciences, Institute of Animal Cell Technology and Systems Biology, Muthgasse 18, 1190 Vienna, Austria.
| | - Giulia Borsi
- BOKU University of Natural Resources and Life Sciences, Institute of Animal Cell Technology and Systems Biology, Muthgasse 18, 1190 Vienna, Austria
| | - Maja Papež
- Austrian Centre of Industrial Biotechnology (acib GmbH), Muthgasse 11, 1190 Vienna, Austria; BOKU University of Natural Resources and Life Sciences, Institute of Animal Cell Technology and Systems Biology, Muthgasse 18, 1190 Vienna, Austria
| | - Caterina Ruggeri
- BOKU University of Natural Resources and Life Sciences, Institute of Animal Cell Technology and Systems Biology, Muthgasse 18, 1190 Vienna, Austria
| | - Martina Baumann
- Austrian Centre of Industrial Biotechnology (acib GmbH), Muthgasse 11, 1190 Vienna, Austria
| | - Nicole Borth
- Austrian Centre of Industrial Biotechnology (acib GmbH), Muthgasse 11, 1190 Vienna, Austria; BOKU University of Natural Resources and Life Sciences, Institute of Animal Cell Technology and Systems Biology, Muthgasse 18, 1190 Vienna, Austria.
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Zhang H, Liang C, Hou X, Wang L, Zhang D. Study of the combined treatment of lung cancer using gene-loaded immunomagnetic albumin nanospheres in vitro and in vivo. Int J Nanomedicine 2016; 11:1039-50. [PMID: 27042059 PMCID: PMC4801199 DOI: 10.2147/ijn.s98519] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Combination therapy for lung cancer has garnered widespread attention. Radiation therapy, gene therapy, and molecular targeted therapy for lung cancer have certain effects, but the disadvantages of these treatment methods are evident. Combining these methods can decrease their side effects and increase their curative effects. In this study, we constructed a pYr-ads-8-5HRE-cfosp-iNOS-IFNG plasmid (a gene circuit that can express IFNγ), which is a gene circuit, and used that plasmid together with C225 (cetuximab) to prepare gene-loaded immunomagnetic albumin nanospheres (IMANS). Moreover, we investigated the therapeutic effects of gene-loaded IMANS in combination with radiation therapy on human lung cancer in vitro and in vivo. The results showed that this gene circuit was successively constructed and confirmed that the expression of INFγ was increased due to the gene circuit. Gene-loaded IMANS combined with radiation therapy demonstrated improved results in vitro and in vivo. In conclusion, gene-loaded IMANS enhanced the efficacy of combination therapy, solved problems related to gene transfer, and specifically targeted lung cancer cells.
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Affiliation(s)
- Hao Zhang
- Department of Imaging and Nuclear Medicine, Medical School of Southeast University, Nanjing, Jiangsu, People's Republic of China
| | - Chen Liang
- Department of Pathology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China
| | - Xinxin Hou
- Department of Pathology, Medical School of Henan Polytechnic University, Jiaozuo, Henan, People's Republic of China
| | - Ling Wang
- Department of Imaging and Nuclear Medicine, Medical School of Southeast University, Nanjing, Jiangsu, People's Republic of China
| | - Dongsheng Zhang
- Jiangsu Key Laboratory for Biomaterials and Devices, Medical School, Southeast University, Nanjing, People's Republic of China
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Gilad AA, Pelled G. New approaches for the neuroimaging of gene expression. Front Integr Neurosci 2015; 9:5. [PMID: 25698946 PMCID: PMC4316713 DOI: 10.3389/fnint.2015.00005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2014] [Accepted: 01/13/2015] [Indexed: 01/19/2023] Open
Affiliation(s)
- Assaf A Gilad
- The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine Baltimore, MD, USA ; Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine Baltimore, MD, USA
| | - Galit Pelled
- The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine Baltimore, MD, USA ; F. M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute Baltimore, MD, USA
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Shi B, Zhang H, Shen Z, Bi J, Dai S. Developing a chitosan supported imidazole Schiff-base for high-efficiency gene delivery. Polym Chem 2013. [DOI: 10.1039/c2py20494k] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Kottmeier K, Weber J, Müller C, Bley T, Büchs J. Asymmetric division ofHansenula polymorphareflected by a drop of light scatter intensity measured in batch microtiter plate cultivations at phosphate limitation. Biotechnol Bioeng 2009; 104:554-61. [DOI: 10.1002/bit.22410] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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Krebs MD, Jeon O, Alsberg E. Localized and sustained delivery of silencing RNA from macroscopic biopolymer hydrogels. J Am Chem Soc 2009; 131:9204-6. [PMID: 19530653 DOI: 10.1021/ja9037615] [Citation(s) in RCA: 134] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The ability to silence the expression of specific genes at a particular location of the body would provide a powerful new therapeutic tool for treatment of diseases such as cancer or for use in regenerative medicine. RNA interference (RNAi) is a gene silencing mechanism where specific mRNA molecules that are complementary to short interfering RNA (siRNA) are degraded, thus inhibiting gene expression at the post-transcriptional level. However, the use of siRNA has not yet realized its full clinical potential due to degradation in vivo, the difficulty retaining siRNA at the site of interest, and the relatively short-term effect it has on rapidly dividing cells. In this work a new paradigm is presented that will allow for the localized delivery of siRNA that is controlled and sustained over time, thus allowing cells at the site of interest to be directly exposed to a gradual release of bioactive siRNA. To accomplish this, three different types of macroscopic, degradable biomaterial hydrogel scaffolds were employed: calcium crosslinked alginate, photocrosslinked alginate, and collagen. Differing rates of release from these hydrogels were achieved, and the ability of the released siRNA to knock down the expression of GFP in cells that constitutively express this protein was shown. Furthermore, the ability to encapsulate cells within these materials and achieve sustained gene silencing of these incorporated cells was demonstrated. These biopolymer hydrogels are injectable and, therefore, can be delivered in a minimally invasive manner, and they can serve as delivery vehicles for both siRNA and transplanted cell populations.
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Affiliation(s)
- Melissa D Krebs
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, USA
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Dennis MK, Bowles HJC, MacKenzie DA, Burchiel SW, Edwards BS, Sklar LA, Prossnitz ER, Thompson TA. A multifunctional androgen receptor screening assay using the high-throughput Hypercyt flow cytometry system. Cytometry A 2008; 73:390-9. [PMID: 18340645 DOI: 10.1002/cyto.a.20552] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen-sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen-independent human prostate cancer-derived PC3 cells were transiently cotransfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low-dose R1881, which also occurred in a dose-dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high-throughput compatibility, the MARS assay and HyperCyt system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.
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Affiliation(s)
- Megan K Dennis
- Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA
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Mattanovich D, Borth N. Applications of cell sorting in biotechnology. Microb Cell Fact 2006; 5:12. [PMID: 16551353 PMCID: PMC1435767 DOI: 10.1186/1475-2859-5-12] [Citation(s) in RCA: 98] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2005] [Accepted: 03/21/2006] [Indexed: 01/28/2023] Open
Abstract
Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology.
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Affiliation(s)
- Diethard Mattanovich
- University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Institute of Applied Microbiology, Muthgasse 18, A-1190 Vienna, Austria
- School of Bioengineering, University of Applied Sciences FH-Campus Vienna, Muthgasse 18, A-1190 Vienna, Austria
| | - Nicole Borth
- University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Institute of Applied Microbiology, Muthgasse 18, A-1190 Vienna, Austria
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Hisiger S, Jolicoeur M. A multiwavelength fluorescence probe: Is one probe capable for on-line monitoring of recombinant protein production and biomass activity? J Biotechnol 2005; 117:325-36. [PMID: 15890426 DOI: 10.1016/j.jbiotec.2005.03.004] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2004] [Revised: 03/16/2005] [Accepted: 03/18/2005] [Indexed: 11/16/2022]
Abstract
Monitoring cell culture performance requires maximizing the number and the quality of measured parameters and in situ 2D fluorescence spectroscopy could allow intensification of simultaneous data acquisition. The use of a multiwavelength fluorescence probe is proposed for monitoring GFP-producing cultures in bioreactor. The yeast Pichia pastoris and NSO mammalian cells were studied as model systems. Tryptophan, NAD(P)H and riboflavins (riboflavin, FMN, FAD) signals were effective for on-line yeast biomass estimation during the growth phase. During the GFP production phase, in situ measurements of the GFP concentration from the fluorescence probe were well correlated with off-line analyses. Tryptophan and NAD(P)H signals diverged from that of biomass during GFP production. With NSO mammalian cells, results showed that the culture parameters have to be optimized for the use of a fluorescence probe. The use of serum and phenol-red interfered with NAD(P)H and riboflavins fluorescence signals. Nevertheless, it appears that a multiwavelength probe could be useful for culture monitoring of biomass, cell activity and recombinant protein expression in an optimized culture medium.
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Affiliation(s)
- Steve Hisiger
- Canada Research Chair on the Development of Metabolic Engineering Tools, Bio-P2 Research Unit, Department of Chemical Engineering, Ecole Polytechnique de Montréal, P.O. Box 6079, Centre-ville Station, Montréal, Quebec, Canada
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Garle MJ, Fry JR. Sensory nerves, neurogenic inflammation and pain: missing components of alternative irritation strategies? A review and a potential strategy. Altern Lab Anim 2005; 31:295-316. [PMID: 15612874 DOI: 10.1177/026119290303100313] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The eyes and skin are highly innervated by sensory nerves; stimulation of these nerves by irritants may give rise to neurogenic inflammation, leading to sensory irritation and pain. Few in vitro models of neurogenic inflammation have been described in conjunction with alternative skin and eye irritation methods, despite the fact that the sensory innervation of these organs is well-documented. To date, alternative approaches to the Draize skin and eye irritation tests have proved largely successful at classifying severe irritants, but are generally poor at discriminating between agents with mild to moderate irritant potential. We propose that the development of in vitro models for the prediction of sensory stimulation will assist in the re-classification of the irritant potential of agents that are under-predicted by current in vitro strategies. This review describes the range of xenobiotics known to cause inflammation and pain through the stimulation of sensory nerves, as well as the endogenous mediators and receptor types that are involved. In particular, it focuses on the vanilloid receptor, its activators and its regulation, as these receptors function as integrators of responses to numerous noxious stimuli. Cell culture models and ex vivo preparations that have the potential to serve as predictors of sensory irritation are also described. In addition, as readily available sensory neuron cell line models are few in number, stem cell lines (with the capacity to differentiate into sensory neurons) are explored. Finally, a preliminary strategy to enable assessment of whether incorporation of a sensory component will enhance the predictive power of current in vitro eye and skin testing strategies is proposed.
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Affiliation(s)
- Michael J Garle
- Division of Gastroenterology, School of Medical and Surgical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK
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Redmond TM, Ren X, Kubish G, Atkins S, Low S, Uhler MD. Microarray transfection analysis of transcriptional regulation by cAMP-dependent protein kinase. Mol Cell Proteomics 2004; 3:770-9. [PMID: 15118071 DOI: 10.1074/mcp.m400018-mcp200] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
A wide variety of bioinformatic tools have been described to characterize potential transcriptional regulatory mechanisms based on genomic sequence analysis and microarray hybridization studies. However, these regulatory mechanisms are still experimentally verified using transient transfection methods. Current transfection methods are limited both by their large scale and by the low level of efficiency for certain cell types. Our goals were to develop a microarray-based transfection method that could be optimized for different cell types and that would be useful in reporter assays of transcriptional regulation. Here we describe a novel transfection method, termed STEP (surface transfection and expression protocol), which employs microarray-based DNA transfection of adherent cells in the functional analysis of transcriptional regulation. In STEP, recombinant proteins with biological activities designed to enhance transfection are complexed with expression vector DNAs prior to spotting on microscope slides. The recombinant proteins used in STEP complexes can be varied to increase the efficiency for different cell types. We demonstrate that STEP efficiently transfects both supercoiled plasmids and PCR-generated linear expression cassettes. A co-transfection assay using effector expression vectors encoding the cAMP-dependent protein kinase (PKA), as well as reporter vectors containing PKA-regulated promoters, showed that STEP transfection allows detection and quantitation of transcriptional regulation by this protein kinase. Because bioinformatic studies often result in the identification of many putative regulatory elements and signaling pathways, this approach should be of utility in high-throughput functional genomic studies of transcriptional regulation.
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Affiliation(s)
- Tanya M Redmond
- Mental Health Research Institute, University of Michigan, Ann Arbor 48109-0669, USA
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Hellweg CE, Baumstark-Khan C, Horneck G. Generation of stably transfected Mammalian cell lines as fluorescent screening assay for NF-kappaB activation-dependent gene expression. ACTA ACUST UNITED AC 2004; 8:511-21. [PMID: 14567778 DOI: 10.1177/1087057103257204] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. To identify conditions that are capable of modifying this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter protein Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) was developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing 4 copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP/d2EGFP expression in up to 90% of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB-dependent gene expression. The time course of NF-kappaB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-kappaB-ELISA. NF-kappaB binding in-creased after 15-min incubation with TNF-alpha. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-kappaB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-alpha to be caused by the activation of NF-kappaB. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.
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Affiliation(s)
- Christine E Hellweg
- Radiation Biology, Institute of Aerospace Medicine, DLR, Linder Höhe, D-51170 Köln, Germany.
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He Q, Liu J, Sun X, Zhang ZR. Preparation and characteristics of DNA-nanoparticles targeting to hepatocarcinoma cells. World J Gastroenterol 2004; 10:660-3. [PMID: 14991933 PMCID: PMC4716904 DOI: 10.3748/wjg.v10.i5.660] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To prepare thymidine kinase gene (TK gene) nanoparticles and to investigate the expression of TK gene.
METHODS: Poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, was used to prepare recombinant plasmid PEGFP-AFP nanoparticles by a double-emulsion evaporation technique. Characteristics of the nanoparticles were investigated in this study, including morphology, entrapment efficiency, and tissue distribution. The expression of TK gene was also investigated by MTT assay, by which the viable cells were determined after the addition of ganciclovir (GCV). The enhanced green fluorescent protein (EGFP) expression in human hepatocellular carcinoma SMMC-7721 cells and normal parenchymal Chang liver cells were assessed by flow cytometry.
RESULTS: The prepared plasmid-nanoparticles had regular spherical surface and narrow particle size span with a mean diameter of 72 ± 12 nm. The mean entrapment efficiency was 91.25%. A total of 80.14% DNA was found to be localized in the livers after 1-h injection with 32P-DNA-PLGA nanoparticles in mouse caudal vein. The expression of DNA encapsulated in nanoparticles was much higher than that in naked DNA, and human hepatocellular carcinoma SMMC-7721 cells were more sensitive to GCV than human normal parenchymal Chang liver cells.
CONCLUSION: The enhanced transfection efficiency and stronger ability to protect plasmid DNA from being degraded by nucleases are due to nanoparticles encapsulation.
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Affiliation(s)
- Qin He
- West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan Province, China.
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Markova SV, Golz S, Frank LA, Kalthof B, Vysotski ES. Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme. J Biol Chem 2003; 279:3212-7. [PMID: 14583604 DOI: 10.1074/jbc.m309639200] [Citation(s) in RCA: 115] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max) = 480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultrahigh throughput screening technologies.
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Affiliation(s)
- Svetlana V Markova
- Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences Siberian Branch, Krasnoyarsk 660036, Russia
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Bi JX, Buhr P, Zeng AP, Wirth M. Human c-fos promoter mediates high-level, inducible expression in various mammalian cell lines. Biotechnol Bioeng 2003; 81:848-54. [PMID: 12557318 DOI: 10.1002/bit.10530] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The promoter activity of the human c-fos and human cytomegalovirus (CMV) immediate early promoter was compared in transient and stable transfection experiments with six cell lines of mouse, human, and hamster origin which are all of commercial importance. The c-fos promoter was 1.8-5.6-fold stronger than the CMV promoter in BHK-A, BHK-B, CHO-DHFR(-), and mouse NIH-3T3 in stable transfectants and less effective in mouse myeloma or human 293 cells, suggesting a new transcriptional control element for high-level expression and protein production in mammalian cells. The induction profiles determined in the presence and absence of serum are dependent on the cell line used. Induction levels of up to 8-fold could be achieved in preselected cell pools.
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Affiliation(s)
- Jing-Xiu Bi
- Biochemical Engineering Division, GBF - German Research Centre for Biotechnology, Braunschweig, D-38124 Germany
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