CMV Promoter-Driven Expression and Visualization of Tagged Proteins in Live and Fixed Zebrafish Embryonic Epidermis

2020 ◽  
pp. 29-41
Author(s):  
Kirti Gupta ◽  
Mahendra Sonawane
Keyword(s):  
Cmv Promoter

scholarly journals The baculovirus promoter OpIE2 sequence has inhibitory effect on the activity of the Cytomegalovirus (CMV) promoter in HeLa and HEK-293T cells

Gene ◽  
2021 ◽  
pp. 145541
Author(s):  
Aladdin ◽  
N. Sahly ◽  
R. Faty ◽  
MM. Youssef ◽  
TZ. Salem
Keyword(s):  
Inhibitory Effect ◽  
Cmv Promoter

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 Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

2012 ◽  
Vol 12 (3) ◽  
pp. 349-356 ◽  
Author(s):  
Daniel Lipiński ◽  
Joanna Zeyland ◽  
Andrzej Pławski ◽  
Ryszard Słomski
Keyword(s):  
Copy Number ◽  
Transgenic Animals ◽  
Promoter Sequence ◽  
Absolute Number ◽  
Transgenic Pigs ◽  
Cmv Promoter ◽  
Genetic Construct ◽  
Coding Sequence ◽  
The Absolute

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic PigsThe aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

scholarly journals Robust cancer-specific gene expression by a novel cassette with hTERT and CMV promoter elements

2017 ◽  
Vol 38 (2) ◽  
pp. 1108-1114
Author(s):  
Masakiyo Sakaguchi ◽  
Takuya Sadahira ◽  
Hideo Ueki ◽  
Rie Kinoshita ◽  
Hitoshi Murata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Gene ◽  
Promoter Elements ◽  
Cmv Promoter ◽  
Specific Gene Expression

scholarly journals Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa

2021 ◽  
Vol 22 (23) ◽  
pp. 12774
Author(s):  
Xianqing Wang ◽  
Fatma Alshehri ◽  
Darío Manzanares ◽  
Yinghao Li ◽  
Zhonglei He ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Gene Replacement ◽  
Epidermal Keratinocytes ◽  
Viral Gene ◽  
Cmv Promoter ◽  
Human Epidermal Keratinocytes ◽  
Dystrophic Epidermolysis Bullosa ◽  
Type Vii Collagen ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Col7a1 Gene

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

Posttreatment with adenovirus-mediated gene transfer of calcitonin gene—related peptide to reverse cerebral vasospasm in dogs

2002 ◽  
Vol 97 (1) ◽  
pp. 136-142 ◽  
Author(s):  
Motoyoshi Satoh ◽  
Eddie Perkins ◽  
Hitoshi Kimura ◽  
Jiping Tang ◽  
Yi Chun ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cerebral Vasospasm ◽  
Treated Group ◽  
Positive Staining ◽  
Cmv Promoter ◽  
Content Type ◽  
Related Peptide ◽  
Arterial Blood ◽  
Blood Injection ◽  
Fine Print

Object. Gene transfer to cerebral vessels is a promising new therapeutic approach for cerebral vasospasm after subarachnoid hemorrhage (SAH). This study was undertaken to explore whether a delayed treatment with adenovirus encoding the prepro-calcitonin gene—related peptide (CGRP), 2 days after initial blood injection, reduces cerebral vasospasm in a double-hemorrhage model of severe vasospasm in dogs. Methods. In 20 dogs, arterial blood was injected into the cisterna magna on Days 0 and 2. Thirty minutes after the second blood injection, the animals received either adenovirus encoding the prepro-CGRP gene (AdCMVCGRP—treated group, eight dogs) or adenovirus encoding the β-galactosidase gene (AdCMVβgal—treated group, six dogs) under the cytomegalovirus (CMV) promoter. One group of dogs did not receive treatment and served as controls (control SAH group, six dogs). Angiography was performed on Days 0 and 7 to assess cerebral vasospasm. On Day 7 following angiography, the animals were killed and their brains were stained with X-gal to detect the distribution of gene expression. Cerebrospinal fluid (CSF) was also tested for CGRP immunoreactivity. Severe vasospasm was observed in control SAH dogs on Day 7, and the mean basilar artery (BA) diameter was 53.4 ± 5.5% of the value measured on Day 0. Treatment with AdCMVβgal did not alter vasospasm (the BA diameter was 55 ± 3.9% of that measured on Day 0). The leptomeninges and adventitia of the BAs of dogs treated using AdCMVβgal demonstrated positive staining with X-gal. High levels of CGRP were measured in CSF from dogs that received AdCMVCGRP. In the group treated with AdCMVCGRP, vasospasm was significantly reduced (the BA diameter was 78.2 ± 5.3% of that measured on Day 0, p < 0.05 compared with the control SAH group and the AdCMVβgal group). Conclusions. In a model of severe vasospasm in dogs, gene transfer of CGRP after injection of blood attenuated cerebral vasospasm after SAH.

scholarly journals Effect of Transcription Factor GATA-2 on Phagocytic Activity of Alveolar Macrophages from Pneumocystis carinii-Infected Hosts

2003 ◽  
Vol 71 (9) ◽  
pp. 4943-4952 ◽  
Author(s):  
Mark E. Lasbury ◽  
Xing Tang ◽  
Pamela J. Durant ◽  
Chao-Hung Lee
Keyword(s):  
Transcription Factor ◽  
Alveolar Macrophages ◽  
Phagocytic Activity ◽  
Pneumocystis Carinii ◽  
Fluorescein Isothiocyanate ◽  
Cmv Promoter ◽  
Latex Beads ◽  
Expression Construct ◽  
Sense Orientation ◽  
General Defect

ABSTRACT Alveolar macrophages from Pneumocystis carinii-infected hosts are defective in phagocytosis (W. Chen, J. W. Mills, and A. G. Harmsen, Int. J. Exp. Pathol. 73:709-720, 1992; H. Koziel et al., J. Clin. Investig. 102:1332-1344, 1998). Experiments were performed to determine whether this defect is specific for P. carinii organisms. The results showed that these macrophages were unable to phagocytose both P. carinii organisms and fluorescein isothiocyanate (FITC)-conjugated latex beads, indicating that alveolar macrophages from P. carinii-infected hosts have a general defect in phagocytosis. To determine whether this defect correlates with the recently discovered down-regulation of the GATA-2 transcription factor gene during P. carinii infection, alveolar macrophages from dexamethasone-suppressed or healthy rats were treated with anti-GATA-2 oligonucleotides and then assayed for phagocytosis. Aliquots of the alveolar macrophages were also treated with the sense oligonucleotides as the control. Cells treated with the antisense oligonucleotides were found to have a 46% reduction in phagocytosis of P. carinii organisms and a 65% reduction in phagocytosis of FITC-latex beads compared to those treated with the sense oligonucleotides. To determine whether the defect in phagocytosis in alveolar macrophages from P. carinii-infected hosts can be corrected by overexpression of GATA-2, a plasmid containing the rat GATA-2 gene in the sense orientation driven by the cytomegalovirus (CMV) promoter was introduced into alveolar macrophages from P. carinii-infected rats. Aliquots of the same cells transfected with a plasmid containing GATA-2 in the antisense orientation relative to the CMV promoter served as the control. Alveolar macrophages treated with the sense GATA-2 expression construct were found to increase their phagocytic activity by 66% in phagocytosis of P. carinii organisms and by 280% in phagocytosis of FITC-latex beads compared to those that received the antisense GATA-2 construct. The results of this study indicate that GATA-2 plays an important role in the regulation of phagocytosis in alveolar macrophages during P. carinii infection.

Rev-binding aptamer and CMV promoter act as decoys to inhibit HIV replication

Gene ◽  
2000 ◽  
Vol 255 (2) ◽  
pp. 235-244 ◽  
Author(s):  
Krystyna Konopka ◽  
Nan Sook Lee ◽  
John Rossi ◽  
Nejat Düzgüneş
Keyword(s):  
Hiv Replication ◽  
Cmv Promoter

304 HUMAN AUGMENTER OF LIVER REGENERATION TRANSGENIC OVINE EMBRYO DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2008 ◽  
Vol 20 (1) ◽  
pp. 232
Author(s):  
Y. Ma ◽  
P. Zhou ◽  
D. Liu ◽  
G. Xia ◽  
S. Bou
Keyword(s):  
Liver Regeneration ◽  
Nuclear Transfer ◽  
Genomic Sequence ◽  
Pcr Amplification ◽  
Foreign Gene ◽  
Green Fluorescence ◽  
Entry Site ◽  
Cmv Promoter ◽  
Coding Sequence ◽  
Augmenter Of Liver Regeneration

The aim of this research was to develop an efficient screening technique to detect transgenic ovine embryos using neomycin resistance (NeoR) and enhanced green fluorescence protein (EGFP) genes as genetic markers. A 0.8-kb fragment of the ovine β-lactoglobulin promoter sequence (BLG) and 1.8 kb of the human augmenter of liver regeneration (ALR) genomic sequence were derived by PCR amplification. These 2 fragments were inserted into the MCS of pGEM-7zf(+) plasmid; this vector was named p7Z-BA. The coding sequence of NeoR was derived by PCR amplification from the plasmid pIRES2-EGFP and was assembled into the MCS of the pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a NeoR gene coding sequence and an EGFP coding sequence linked by an internal ribosome entry site (IRES) sequence downstream of the CMV promoter. The vector pNIE was excised as an NsiI-SspI fragment and inserted into the vector p7Z-BA. In the end, we had a vector named pNEA, which contained the NeoR gene and the EGFP gene regulated by a CMV promoter for expression in a non-tissue specific mode, and the human ALR gene regulated by the BLG promoter for expression specifically in mammary gland. Sheep fetal fibroblast (SFFB) cells were isolated by attachment of tissue pieces from the ear skin of a 1- to 2-month ovine fetus. Karyotypes of the cells at the third passage and after 15 passages were analyzed. The cells proliferated well and more than 72% of the cells maintained a diploid karyotype after 15 passages. Therefore, the SFFB cells are amenable for transgenic cloning manipulations. For transfection, third-passage SFFB cells at 70% confluency were transfected in a 100-mm dish with pNEA (0.5, 1.0, 2.0, 3.0, 5.0, and 7.0 µg) using Lipofectamine 2000 (2, 4, 6, 8, 10, and 12 µL; Invitrogen, Carlsbad, CA). Cells were checked 24 to 48 h after transfection under fluorescence microscopy for GFP expression, and G418 selection (800 µg mL–1) was applied at that time. After 2 weeks, selected colonies were counted and propagated in culture medium containing 300 µg mL–1 G418 for 2 to 3 passages and cryopreserved. A small portion of the cells was analyzed by PCR for gene integration. Bright green fluorescence could be detected 24 to 48 h after transfection. More colonies were selected when transfection parameters were 2 µg of DNA and 10 µL of Lipofectamine. The results of PCR detection showed that the foreign gene was integrated into the genome. A total of 612 oocytes were aspirated from 2- to 5-mm follicles of ovine ovaries collected from an abattoir; 78% of them were matured after 18 h in culture. Four hundred forty-three oocytes were enucleated, and 332 enucleated oocytes were treated for electrofusion with green fluorescence cells. Of these, 180 (54.2%) couplets were fused. A total of 172 reconstructed embryos were stimulated and cultured in vitro, 31 (18%) of which developed to the blastocyst stage, and 19 blastocysts expressed GFP. In conclusion, we established an effective method to select transgenic embryos formed by nuclear transfer using transfected donor cells.

Percutaneous in vivo gene transfer to the peripheral lungs using plasmid-liposome complexes

2000 ◽  
Vol 279 (4) ◽  
pp. L651-L657 ◽  
Author(s):  
Hiroshi Saito ◽  
Hidenori Nakamura ◽  
Suichi Kato ◽  
Sumito Inoue ◽  
Minoru Inage ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Lung Parenchyma ◽  
Firefly Luciferase ◽  
Local Area ◽  
Cationic Lipids ◽  
Limited Area ◽  
Cmv Promoter ◽  
Percutaneous Injection ◽  
In Vivo Gene Transfer

The purpose of this study was to investigate a new method of in vivo gene transfer to the lung parenchyma by the percutaneous approach. The plasmid that contains the gene for firefly luciferase driven by a cytomegalovirus (CMV) promoter (pCMVL) in combination with cationic lipids was percutaneously injected into the lung parenchyma. Luciferase activities were localized to the lobes of the lung where the plasmids with cationic lipids were injected. Percutaneous injection of the plasmid containing the human endothelin-1 (hET-1) gene driven by a CMV promoter (pRc/CMVhET-1) in combination with cationic lipids into the lungs caused pulmonary fibrosis localized to the injection site in the peripheral lungs. We concluded that percutaneous in vivo gene transfer to the lungs is a unique and important approach to introduce exogenous gene expression in the limited area of the lung parenchyma. This method of gene transfer will be applicable for human gene therapy for targeted areas of peripheral lung and will also be useful to assess the function of the proteins expressed by a gene in the local area of the lungs.

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