Why use cmv promoter
No statistical significance was observed for the difference in FLK1 expression in cells transfected with the different constructs. Transfected cells were selected for puromycin resistance and the cells were differentiated on collagen type IV for 5 days. FACS analysis confirmed that high levels of cell surface expression of both proteins was achieved in undifferentiated as well as differentiated cells Fig. The CAG promoter provides long term expression of transgenes in these cells however the system is most effective when the cells can be put under a selective pressure to express the transgene.
This presents a very useful tool for use in studies of angiogenesis and vasculogenesis. The polyclonal chicken anti-syndecan-4 antibody Harlan Sera-Lab, UK was raised against the N-terminal 20 amino acids of the mature human protein.
Antibodies were affinity purified from plasma using standard procedures. For exclusion of dead cells, the nucleic acid dye Topro-3 0. Cells stained with secondary antibody only or empty vector transfected cells served as controls for background fluorescence. For FACS sorting, dead cells were excluded by propidium iodide staining 0. Controls included staining with secondary antibody only, while in double labelling experiments, controls included incubation with one primary antibody and both secondary antibodies.
Inappropriate cross-reactivity was minimal. Micrographs were taken on an Olympus DP50 microscope and digital camera system. Images were processed using the Viewfinder Lite software and Adobe Photoshop 7.
Three dimensional collagen type I cultures were fixed with 3. Fixation with paraformaldehyde was repeated for 1 h at room temperature. Excess gelatin was removed and samples were stored in 0. Sections were permeabilised with 0. Sections were washed with PBS, then incubated in secondary antibody for 5 h at room temperature. After further extensive washing, sections were mounted onto slides using Gene Frame gaskets Advanced Biotechnologies and were sealed with coverslips.
OligodT was used to initiate cDNA synthesis and the syndecan gene specific primers are as detailed in Table 2. Huxley [ 17 ], Imperial College London. For transfection in suspension, , CCE cells were plated per well of a six-well plate 24 h prior to transfection. Cells were trypsinised and washed in serum free DMEM. Fresh medium was added after 24 h.
Fibroblasts were transfected with lipofectamine following standard procedures. Physiol Rev. Feraud O, Cao Y, Vittet D: Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis. Lab Invest. Rejuvenation Res. Article Google Scholar. Stem Cells. Article PubMed Google Scholar. Ma H, Liu Q, Diamond SL, Perce EA: Mouse embryonic stem cells efficiently lipofected with nuclear localisation peptide result in a high yield of chimeric mice and retain germline transmission potency.
Biotechnol Lett. Ward CM, Stern PL: The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells. Stem cells. Suzuki H, Watabe T, Kato M, Miyazawa K, Miyazono K: Roles of vascular endothelial growth factor receptor 3 signaling in differentiation of mouse embryonic stem cell-derived vascular progenitor cells into endothelial cells. Methods Enzymol. J Histochem Cytochem. Redick SD, Bautch VL: Developmental platelet endothelial cell adhesion molecule expression suggests multiple roles for a vascular adhesion molecule.
Am J Pathol. Int J Biochme Cell Biol. Arterioscler Thromb Vasc Biol. J Clin Invest. Fears CY, Gladson CL, Woods A: Syndecan-2 is expressed in the microvasculature of gliomas and regulates angiogenic processes in microvascular endothelial cells. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters.
We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Correspondence and Footnotes. Luciferase assays of transfected cells untreated or treated with IFN-g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines.
During the past few years, the use of mammalian gene expression vectors for new methods such as genetic immunization has become increasingly important.
Recent success with DNA vaccines indicates their enormous future potential in diverse fields such as infectious diseases, allergy and cancer Depending on the antigen processing pathway, proteins produced through mammalian expression vectors can stimulate humoral or cellular immunity.
Intracellular expression of a non-secreted antigen should specifically induce cellular immunity through presentation by the major histocompatibility complex class I MHC I molecules to cytotoxic T cells. During the immune response, many lymphokines are produced by the various cells involved, including IFN-g, that can act on the target cell carrying the transgene. Modulation of expression of the transfected gene could occur and, depending on the promoter used, increase or decrease transgene expression.
Although these promoter elements are from pathogenic viruses, they have become very useful due to high transcription initiation ability in most mammalian tissues 8. However, in genetic immunization applications involving human patients or animal husbandry, a mammalian promoter may be more desirable for easing public concern over transfecting DNA elements from pathogenic or tumor-causing viruses.
BLprmtr is the promoter for the cattle major histocompatibility complex class I gene. MHC I is expressed on nearly all tissues, is critical in immune system communication, and can be up-regulated by lymphokines such as IFN-g 9. Additionally, all three promoters have interferon response sequences.
However, for CMV and SV40, factors binding to this region can inhibit transcription 13,14 , whereas, for MHC I, interferon response factors enhance transcription 15, Each of the three promoters was able to drive luciferase expression in all cell lines tested. Luciferase production was positively or negatively altered by IFN-g treatment depending on the promoter and cell line. Transiently transfected cell cultures were treated or not treated with rIFN-g for 16 h and then assayed for luciferase production.
Results demonstrate that BLprmtr-driven luciferase production increased in all cell lines, whereas the SV40 or CMV promoter-driven luciferase production decreased in most cell lines Table 2. We isolated neomycin-resistant clones of A J, P, RAW Many gene delivery vaccination applications use muscle as the target tissue Figure 1 gives a graphic illustration of this change. Three experiments of three transfections of each luciferase construct were performed. The kinetics of luciferase transgene production showed that the amount of CMV-driven luciferase product peaked at approximately 8 h and then dropped to below baseline untreated levels at h, while the MHC I-driven luciferase product continued to rise through 48 h.
Figure 2 - Kinetics of luciferase transgene expression in stably transfected cell lines. Graphs show the mean relative light units RLU and standard deviations of four experiments.
An in vivo immune response that was either initiated or promoted by the transgene would release a multitude of lymphokines, along with IFN-g, that may have additive, synergistic, or antagonistic effects on the transgene promoter. Using the supernatant from mitogen-stimulated T cells as a source of immune response lymphokines, we tested transgene expression in response to these mixed lymphokines as well as IFN-g in a transiently transfected human colorectal carcinoma cell line.
The results, shown in Figure 3 , indicate that the T cell blast supernatant increased transgene expression driven by MHC I promoter to an even greater extent than IFN-g alone.
Viral promoter-driven expression was up-regulated by the mixed lymphokines compared to IFN-g-treated cells but not to the level of untreated cells. This choice is dependent upon target cell type as well as the functional goal of the transgene.
DNA vaccine applications are designed to affect immune function to protect against a pathogenic agent, or lymphokine gene transfer to fight cancer. These applications stimulate the immune system resulting in production of many lymphokines that can directly act on the transfected target cell and thus indirectly act on the transgene promoter.
Nonetheless, these negative selective pressures may shorten the expressible life of the transgene. For example, we noticed in our stably transfected cell clones that luciferase expression driven by BLprmtr stayed at the same level for many cell passages whereas luciferase expression driven by CMV promoter showed a "dampening" of expression soon after cloning.
This theory has yet to be tested. In fact, studies using the stable transfects showed an ever increasing amount of luciferase from MHC I promoter-driven transgene over h while amounts of luciferase from transfect cultures of CMV promoter-driven transgene were near or below baseline levels at this time. These experiments showed an initial increase in CMV promoter-driven production peaking at around 8 h and then dropping to below baseline levels. The promoter is a major element in the expression cassette of gene therapy vectors, and optimal promoter selection can increase target specificity and gene expression Several reports investigated the effect of promoters on transgene expression 35 , At present, CMV is the most commonly used promoter for the production of recombinant protein However, the CMV promoter cannot maintain production stability over time and it has many potential methylation sites; mutations and methylation will lead to lower productivity of recombinant proteins However, we found that the CAG promoter cannot enhance transgene expression in long-term culture.
This phenomenon may result from methylation of the CAG promoter In this study, the CMV mutant was unable to improve exogenous gene expression when the cytosine at position was point-mutated to guanine; hence, we speculate that CG point mutations may inhibit transgene expression.
During the long-term passage of cells, non-viral-mediated vectors that contain large DNA fragments can cause deficient transgene expression.
These results demonstrated that there was no direct relationship between exogenous gene expression levels and gene copy number. These results are consistent with the results of eGFP expression. Many studies investigated synthetic promoters However, human CMV usually peaks 1—2 days after transfection and the activity is rapidly lost The synthetic promoter would shorten the vector length and potentially contribute to the improvement of transfection efficiency.
However, this experiment was performed only in CHO cells and the results cannot be extrapolated to other cell lines. The development of such expression systems is a major strategic task continually required for the expression of target proteins for research and is necessary for any future long-term clinical application.
Our study makes a significant contribution to such research and applications. The sequences of elements were synthesized by General Biosystems Chuzhou, China. The EPO gene was synthesized according to the codon optimization sequence and further cloned into the vector using standard methods Schematic representation of vectors containing different promters and the length of different elements.
A The vectors construction that containing different element. Each cell type was plated into three wells. The MFI and transfection efficiency were detected. The numbers of eGFP-positive and eGFP-negative cells were calculated according to the flow cytometry results, and the transfection efficiency was represented by the ratio of the number of eGFP-positive cells to the total cell number.
Long-term stable expression of the target gene is required for industrial production of recombinant proteins. This software provides values for the media fluorescence intensity MFI based on the fluorochromes in each cell captured by flow cytometry. Each ten passages, the expression level of eGFP was determined by fluorescence intensity. Oligonucleotide primer sequences are provided in Table 1.
Copy numbers were determined in triplicate and are presented as ratios of individual copy numbers relative to the control. After approximately 10 passages, the expression levels of recombinant mRNA were analyzed.
The mRNA expression level of the target gene was calculated by comparison with that of the internal reference gene. Colonies arose after 10—14 days. Briefly, like the method demonstrated in stable expression of transfected cells and flow cytometry analysis. The cells transfected with EPO-containing vector were suspended. Densitometric analysis was performed by using ImageJ v2.
All experimental data were analyzed by using SPSS All experiments were performed three times and t-tests were used for comparisons. Hoban, M. Genetic treatment of a molecular disorder: gene therapy approaches to sickle cell disease. Garcia-Gomez, M. Flynn, R. Wang, T. Cell compatibility of an eposimal vector mediated by the characteristic motifs of matrix attachment regions.
Gene Ther. Hansen, H. Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: lessons learned and future directions. Walsh, G. Biopharmaceutical benchmarks. Wang, X. Santilli, G. Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells. Stein, S. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.
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Rotondaro, L. Gene , — Addison, C. Comparison of the human versus murine cytomegalovirus immediate early gene promoters for transgene expression by adenoviral vectors. Rita Costa, A. Guidelines to cell engineering for monoclonal antibody production.
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