scholarly journals Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo

Gene Therapy ◽  
2000 ◽  
Vol 7 (7) ◽  
pp. 556-567 ◽  
Author(s):  
D S Steinwaerder ◽  
A Lieber
Keyword(s):  
Transgene Expression ◽  
Regulatory Elements ◽  
Transcriptional Regulatory Elements ◽  
Transcriptional Regulatory ◽  
Adenovirus Vectors

scholarly journals Long-Term Transgene Expression in Proliferating Cells Mediated by Episomally Maintained High-Capacity Adenovirus Vectors

2004 ◽  
Vol 78 (1) ◽  
pp. 9-22 ◽  
Author(s):  
Florian Kreppel ◽  
Stefan Kochanek
Keyword(s):  
Transgene Expression ◽  
High Capacity ◽  
Target Cells ◽  
Resting Cells ◽  
Proliferating Cells ◽  
Daughter Cells ◽  
Adenovirus Vectors

ABSTRACT High-capacity “gutless” adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

Strength evaluation of transcriptional regulatory elements for transgene expression by adenovirus vector

2002 ◽  
Vol 81 (1-2) ◽  
pp. 155-163 ◽  
Author(s):  
Zhi-Li Xu ◽  
Hiroyuki Mizuguchi ◽  
Akiko Ishii-Watabe ◽  
Eriko Uchida ◽  
Tadanori Mayumi ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Adenovirus Vector ◽  
Regulatory Elements ◽  
Strength Evaluation ◽  
Transcriptional Regulatory Elements ◽  
Transcriptional Regulatory

scholarly journals Unbiased, Genome-Wide In Vivo Mapping of Transcriptional Regulatory Elements Reveals Sex Differences in Chromatin Structure Associated with Sex-Specific Liver Gene Expression

2010 ◽  
Vol 30 (23) ◽  
pp. 5531-5544 ◽  
Author(s):  
Guoyu Ling ◽  
Aarathi Sugathan ◽  
Tali Mazor ◽  
Ernest Fraenkel ◽  
David J. Waxman
Keyword(s):  
High Throughput Sequencing ◽  
Strong Association ◽  
Regulatory Elements ◽  
Sequence Motifs ◽  
Transcriptional Regulatory Elements ◽  
Transcriptional Regulatory ◽  
Mammalian Tissues ◽  
Hypersensitive Sites ◽  
Regulatory Sites

ABSTRACT We have used a simple and efficient method to identify condition-specific transcriptional regulatory sites in vivo to help elucidate the molecular basis of sex-related differences in transcription, which are widespread in mammalian tissues and affect normal physiology, drug response, inflammation, and disease. To systematically uncover transcriptional regulators responsible for these differences, we used DNase hypersensitivity analysis coupled with high-throughput sequencing to produce condition-specific maps of regulatory sites in male and female mouse livers and in livers of male mice feminized by continuous infusion of growth hormone (GH). We identified 71,264 hypersensitive sites, with 1,284 showing robust sex-related differences. Continuous GH infusion suppressed the vast majority of male-specific sites and induced a subset of female-specific sites in male livers. We also identified broad genomic regions (up to ∼100 kb) showing sex-dependent hypersensitivity and similar patterns of GH responses. We found a strong association of sex-specific sites with sex-specific transcription; however, a majority of sex-specific sites were >100 kb from sex-specific genes. By analyzing sequence motifs within regulatory regions, we identified two known regulators of liver sexual dimorphism and several new candidates for further investigation. This approach can readily be applied to mapping condition-specific regulatory sites in mammalian tissues under a wide variety of physiological conditions.

scholarly journals The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo

2021 ◽  
Author(s):  
Julian Stolper ◽  
Holly K. Voges ◽  
Michael See ◽  
Neda Rahmani Mehdiabadi ◽  
Gulrez Chahal ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Regulatory Element ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Homeodomain Protein ◽  
Cardiovascular Development ◽  
Vascular Expression

AbstractThere is growing evidence that mutations in non-coding cis-regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1. We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer (ΔenFLT1) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.

scholarly journals Possibility for Transcriptional Targeting of Cancer-Associated Fibroblasts—Limitations and Opportunities

2021 ◽  
Vol 22 (7) ◽  
pp. 3298
Author(s):  
Dina V. Antonova ◽  
Marina V. Zinovyeva ◽  
Liya G. Kondratyeva ◽  
Alexander V. Sass ◽  
Irina V. Alekseenko ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Cancer Associated Fibroblasts ◽  
Transcriptional Targeting ◽  
Viral Promoters ◽  
Therapeutic Molecules ◽  
Selective Activity

Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

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