New function of a well‐known promoter: enhancer activity of minimal CMV promoter enables efficient dual‐cassette transgene expression

2021 ◽  
Author(s):  
Michael K. A. Boateng‐Antwi ◽  
Yi Lin ◽  
Sheng Ren ◽  
Xiaohong Wang ◽  
Dao Pan
Keyword(s):  
Transgene Expression ◽  
Cmv Promoter ◽  
Enhancer Activity

scholarly journals Evaluation of Transduction Properties of an Adenovirus Vector in Neonatal Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Shunsuke Iizuka ◽  
Fuminori Sakurai ◽  
Kahori Shimizu ◽  
Kazuo Ohashi ◽  
Shin-ichiro Nakamura ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Adenovirus Vector ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Cmv Promoter ◽  
Neonatal Mice ◽  
Neonatal Heart ◽  
Neonatal Liver ◽  
Neonates And Infants

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of lifeviaretro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.

scholarly journals A transgenic pig model expressing a ZsGreen1 reporter across an extensive array of tissues

2018 ◽  
Author(s):  
Amy T. Desaulniers ◽  
Rebecca A. Cederberg ◽  
Elizabeth P. Carreiro ◽  
Channabasavaiah B. Gurumurthy ◽  
Brett R. White
Keyword(s):  
Transgene Expression ◽  
Specific Activity ◽  
Integration Site ◽  
Expression Patterns ◽  
Genetically Engineered ◽  
Cmv Promoter ◽  
Tissue Samples ◽  
Tissue Specific ◽  
Wide Range ◽  
Agricultural Industries

ABSTRACTBackgroundThe advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene with a single integration site were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot.ResultsZsGreen1 was detected in all tissues examined; however, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within organs of the digestive system, for example, ZsGreen1 was most abundant in the salivary gland, moderately produced in the esophagus and levels were lowest in the stomach. Interestingly, abundance of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. There was no effect of transgene dose as ZsGreen1 expression patterns were similar between homozygous and hemizygous piglets.ConclusionsUltimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig. Moreover, this model can serve as a useful tool for research applications requiring reporter gene activity in mammalian organs.

scholarly journals Regulation of Adenovirus-Mediated Transgene Expression by the Viral E4 Gene Products: Requirement for E4 ORF3

1999 ◽  
Vol 73 (10) ◽  
pp. 8308-8319 ◽  
Author(s):  
M. Lusky ◽  
L. Grave ◽  
A. Dieterlé ◽  
D. Dreyer ◽  
M. Christ ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Transgene Expression ◽  
Cystic Fibrosis Gene ◽  
Gene Products ◽  
In Vivo Evaluation ◽  
Cmv Promoter ◽  
Reading Frame ◽  
E4 Region

ABSTRACT In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022–2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

scholarly journals Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

2019 ◽  
Author(s):  
Binhui Zhao ◽  
Pankaj Chaturvedi ◽  
David L. Zimmerman ◽  
Andrew S. Belmont
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Large Fraction ◽  
Fine Tuning ◽  
Cmv Promoter ◽  
Chromosome Position ◽  
High Level ◽  
Genomic Regions ◽  
Expression In Mammalian Cells

ABSTRACTAchieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouseMsh3-Dhfrlocus (DHFR BAC). Here we extend this “BAC TG-EMBED” approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.

scholarly journals Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1448-1457 ◽  
Author(s):  
Fang Zhang ◽  
Susannah I. Thornhill ◽  
Steven J. Howe ◽  
Meera Ulaganathan ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Lentiviral Vector ◽  
Severe Combined Immunodeficiency ◽  
Insertion Site ◽  
Housekeeping Genes ◽  
Enhancer Activity ◽  
Position Effects ◽  
Chromatin Opening

AbstractUbiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter

2012 ◽  
Vol 78 (4) ◽  
pp. 793-802 ◽  
Author(s):  
B. Duan ◽  
L. Cheng ◽  
Y. Gao ◽  
F.X. Yin ◽  
G.H. Su ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Cmv Promoter

scholarly journals EXTH-41. A NANOPARTICLE-BASED CANCER-SELECTIVE GENE TRANSFER STRATEGY FOR LOCALIZED THERAPY OF MALIGNANT HIGH GRADE GLIOMAS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi90-vi91
Author(s):  
Divya Rao ◽  
Ashish Phal ◽  
Varun Naga ◽  
Karina Negron ◽  
Yumin Oh ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cancer Cells ◽  
Transgene Expression ◽  
Human Cancer ◽  
High Grade ◽  
Cmv Promoter ◽  
Survivin Promoter ◽  
Dna Nanoparticles ◽  
Tumor Tissues ◽  
High Grade Gliomas

Abstract Treatment for malignant high grade gliomas entails surgery, followed by chemotherapy and/or radiation. Despite the intensive care, there is a ~90% recurrence rate. This is attributed to suboptimal efficacy of treatments in tackling widespread cancer cells. We have previously developed nanoparticles that provide widespread therapeutic distribution both in healthy and tumor tissues, but are incapable of differentiating between them. This limitation applies to the current standard-of-care and state-of-the-art gene therapies. Here, we evaluate the efficacy of DNA nanoparticles carrying promoters that drive transgene expression specifically in tumor cells to achieve widespread yet cancer-selective gene transfer in high grade gliomas. To identify tumor-specific promoters, we used ELISA to confirm elevated expression of proteins previously reported to be upregulated in tumor tissue. We observed that expression of survivin in cancer cells was significantly greater than that of other cancer-rich proteins, exhibiting two orders of magnitude greater levels in rodent and human cancer cells compared to their respective healthy cells. Furthermore, the CMV promoter mediated similarly high expression in healthy cells, whereas the level achieved by the survivin promoter was significantly lower, if not negligible, suggesting its tumor specificity. Likewise, CMV-driven plasmids delivered into the brain by the nanoparticles mediated virtually identical volumetric distribution of transgene expression in both normal and tumor tissues in vivo. In contrast, nanoparticles carrying survivin-driven plasmids provided widespread transgene expression only in an orthotopically established tumor, but not healthy brain tissue. Additionally, we demonstrate therapeutic efficacy in an established brain tumor model using the DNA nanoparticles carrying survivin promoter-driven plasmids expressing a therapeutic protein. We identified survivin promoter as a lead TSP and confirmed its ability to mediate highly efficient and widespread but cancer-selective transgene expression with the aid of our nanoparticles uniquely designed to penetrate in healthy and tumor tissues.

scholarly journals RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Wen Wang ◽  
Yan-long Jia ◽  
Yi-chun Li ◽  
Chang-qin Jing ◽  
Xiao Guo ◽  
...  
Keyword(s):  
Cho Cells ◽  
Transgene Expression ◽  
Recombinant Protein Expression ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Vector System ◽  
Gene Copy ◽  
Elongation Factor 1Α ◽  
Cmv Promoter ◽  
Expression Levels

Abstract In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Clonal Marking of Mouse Hematopoietic Stem and Lymphoid Progenitor Cell Populations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5544-5544
Author(s):  
Mariluz P. Mojica-Henshaw ◽  
L. Jeanne Pierce ◽  
John D. Phillips ◽  
Vicente Planelles ◽  
Gerald J. Spangrude
Keyword(s):  
Bone Marrow ◽  
Transgene Expression ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Random Sequence ◽  
Transduction Efficiency ◽  
Cell Populations ◽  
Lentivirus Vector ◽  
Cmv Promoter ◽  
Hematopoietic Stem

Abstract We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1lowc-kithigh) or progenitor cell (Thy-1.1negc-kithigh) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1negc-kithigh progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1lowc-kithigh HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.

Sign in / Sign up

Export Citation Format

Share Document