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Mark A. Kay - One of the best experts on this subject based on the ideXlab platform.
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a mini intronic plasmid mip a novel robust transgene Expression vector in vivo and in vitro
Molecular Therapy, 2013Co-Authors: Feijie Zhang, Mark A. KayAbstract:The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene Expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene Expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene Expression Cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic Expression Cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene Expression compared with minicircle vectors containing the same Expression Cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.
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the extragenic spacer length between the 5 and 3 ends of the transgene Expression Cassette affects transgene silencing from plasmid based vectors
Molecular Therapy, 2012Co-Authors: Feijie Zhang, Andrew Fire, Mark A. KayAbstract:In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene Expression compared to that achieved with a canonical plasmid containing the same Expression Cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5′ and 3′ ends of the transgene Expression Cassette and determined the Expression profiles using two different reporter Expression Cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene Expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3′-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the Expression Cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.
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The Extragenic Spacer Length Between the 5′ and 3′ Ends of the Transgene Expression Cassette Affects Transgene Silencing From Plasmid-based Vectors
Molecular therapy : the journal of the American Society of Gene Therapy, 2012Co-Authors: Feijie Zhang, Andrew Fire, Mark A. KayAbstract:In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene Expression compared to that achieved with a canonical plasmid containing the same Expression Cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5′ and 3′ ends of the transgene Expression Cassette and determined the Expression profiles using two different reporter Expression Cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene Expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3′-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the Expression Cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.
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improved production and purification of minicircle dna vector free of plasmid bacterial sequences and capable of persistent transgene Expression in vivo
Human Gene Therapy, 2005Co-Authors: Zhiying Chen, Mark A. KayAbstract:We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene Expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage oC31 integrase gene and a therapeutic Expression Cassette flanked with attB and attP sites. The oC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic Expression Cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX Expression Cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene Expression make these vectors viable for gene therapy applications.
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Increased Maintenance and Persistence of Transgenes by Excision of Expression Cassettes from Plasmid Sequences In Vivo
Human gene therapy, 2005Co-Authors: Efren Riu, Dirk Grimm, Zan Huang, Mark A. KayAbstract:Persistence of transgene Expression is a major limitation for nonvirus-mediated gene therapy approaches. We have suggested that covalent linkage of bacterial DNA to the Expression Cassette plays a critical role in transcriptional silencing of transgenes in vivo. To gain insight into the role of the covalent linkage of plasmid DNA to the Expression Cassette and transcriptional repression, and whether this silencing effect could be alleviated by altering the molecular structure of vector DNAs in vivo, we generated a scheme for converting routine plasmids into a purified Expression Cassette, free of bacterial DNA after gene transfer in vivo. To do this, the human � -1-antitrypsin (hAAT) and human clotting factor IX (hfIX) reporter genes were flanked by two ISceI endonuclease recognition sites, and coinjected together with a plasmid encoding the I-SceI cDNA or a control plasmid into mouse liver. Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed low serum levels of hAAT or hFIX, which remained low throughout the length of the experiment. However, animals that expressed I-SceI had a 5- to 10-fold increase in serum hAAT or hFIX that persisted for at least 8 months (length of study). Expression of I-SceI resulted in cleavage and excision of the Expression Cassettes from the plasmid backbone, forming mostly circles devoid of bacterial DNA sequences, as established by a battery of different Southern blot and polymerase chain reaction analyses in both C57BL/6 and scid treated mice. In contrast, only the input parental circular plasmid DNA band was detected in mice injected with the reporter gene alone, or an I-SceI plasmid together with the hAAT reporter plasmid lacking the I-SceI sites. Similar results were obtained when the Flp recombinase system was used to make mini-plasmids in mouse liver in vivo. This study presents further independent evidence that removing the covalent linkage between plasmid and transgene sequences leads to a marked increase in and persistence of transgene Expression. Unraveling the mechanisms by which the covalent linkage of bacterial DNA to the Expression Cassette is connected to gene silencing is fundamental to establishing the mechanism of transcriptional regulation in mammalian systems and will be important for the development of versatile nonviral vectors that can be used to achieve persistent gene Expression in different cell types.
Feijie Zhang - One of the best experts on this subject based on the ideXlab platform.
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a mini intronic plasmid mip a novel robust transgene Expression vector in vivo and in vitro
Molecular Therapy, 2013Co-Authors: Feijie Zhang, Mark A. KayAbstract:The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene Expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene Expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene Expression Cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic Expression Cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene Expression compared with minicircle vectors containing the same Expression Cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.
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the extragenic spacer length between the 5 and 3 ends of the transgene Expression Cassette affects transgene silencing from plasmid based vectors
Molecular Therapy, 2012Co-Authors: Feijie Zhang, Andrew Fire, Mark A. KayAbstract:In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene Expression compared to that achieved with a canonical plasmid containing the same Expression Cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5′ and 3′ ends of the transgene Expression Cassette and determined the Expression profiles using two different reporter Expression Cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene Expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3′-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the Expression Cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.
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The Extragenic Spacer Length Between the 5′ and 3′ Ends of the Transgene Expression Cassette Affects Transgene Silencing From Plasmid-based Vectors
Molecular therapy : the journal of the American Society of Gene Therapy, 2012Co-Authors: Feijie Zhang, Andrew Fire, Mark A. KayAbstract:In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene Expression compared to that achieved with a canonical plasmid containing the same Expression Cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5′ and 3′ ends of the transgene Expression Cassette and determined the Expression profiles using two different reporter Expression Cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene Expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3′-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the Expression Cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.
Kostas Iatrou - One of the best experts on this subject based on the ideXlab platform.
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High-level Expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel Expression vector.
Biotechnology and Bioengineering, 1998Co-Authors: Patrick J. Farrell, Maolong Lu, Jay M. Prevost, Christopher B. Brown, Leo A Behie, Kostas IatrouAbstract:: An Expression Cassette for continuous high-level Expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell Expression systems. The Expression Cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive Expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene Expression. Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures. A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells. A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant Expression Cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system. Finally, we show that recombinant protein Expression levels in transformed Bm5 cells remain high in serum-free media, that Expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new Expression Cassette for producing recombinant proteins.
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a baculovirus bombyx mori nuclear polyhedrosis virus repeat element functions as a powerful constitutive enhancer in transfected insect cells
Journal of Biological Chemistry, 1997Co-Authors: Patrick J. Farrell, Russell R. Johnson, Kostas IatrouAbstract:Abstract It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the Expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based Expression Cassette results in an augmentation of transgene Expression in transfected cells by two orders of magnitude relative to the control recombinant Expression Cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the Expression Cassette and occurs only when the HR3 element is linked to the Expression Cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of Expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell Expression system for continuous high-level Expression of recombinant proteins. Such a system should provide levels of Expression of recombinant proteins comparable to those obtained from baculovirus Expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of Expression of proteins from genomic as well as cDNA sequences.
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A cellular promoter-based Expression Cassette for generating recombinant baculoviruses directing rapid Expression of passenger genes in infected insects.
Virology, 1992Co-Authors: Russell R. Johnson, Roy G. Meidinger, Kostas IatrouAbstract:We have developed an Expression Cassette which allows the generation of recombinant baculoviruses that can express passenger genes under the control of a constitutive cellular promoter derived from the cytoplasmic actin gene of the silkmoth Bombyx mori. Silkmoth tissue culture cells which were infected with a recombinant B. mori nuclear polyhedrosis virus (BmNPV) containing the gene-encoding chloramphenicol acetyltransferase (CAT) under the control of this Expression Cassette expressed significant CAT activity beginning 5 hr postinfection (p.i.). Cells infected with a recombinant BmNPV containing the cat gene under the control of the polyhedrin gene promoter did not express CAT activity until 20 hr p.i. Silkworm larvae were also infected with the two recombinant viruses by hemocelic injections and all larval tissues examined were found to express the cat gene. While significant actin-Cassette-driven CAT Expression in vivo was first seen at 24 hr p.i., Expression from the polyhedrin promoter was not seen until 48 hr p.i. By 60 hr p.i., tissues of larvae infected with the recombinant virus expressing cat under polyhedrin promoter control were found to exhibit sixfold higher CAT activity than those infected with recombinant virus expressing the cat gene under the control of the actin promoter. The 24-hr temporal advantage in Expression of a passenger gene in infected larvae indicates that the actin-promoter-based Expression Cassette or other analogous cellular promoter-based Cassettes could be used for generating recombinant baculovirus insecticides which could incapacitate pest insects more quickly than viruses employing the polyhedrin or other late viral promoters for expressing insect-incapacitating proteins.
Helene Faustrup Kildegaard - One of the best experts on this subject based on the ideXlab platform.
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Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering
ACS Synthetic Biology, 2019Co-Authors: Nuša Pristovšek, Saranya Nallapareddy, Lise Marie Grav, Hooman Hefzi, Peter Rugbjerg, Henning Gram Hansen, Mikael Rordam Andersen, Nathan E Lewis, Helene Faustrup KildegaardAbstract:Many branches of biology depend on stable and predictable recombinant gene Expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated Expression Cassette. Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the Expression Cassette components. Here we show in a systematic manner how multiple components contribute to final net Expression of recombinant genes in a characterized integration site. We develop a CRISPR/Cas9-based toolbox for construction of mammalian cell lines with targeted integration of a landing pad, containing a recombinant gene under defined 5′ proximal regulatory elements. Generated site-specific recombinant cell lines can be used in a streamlined recombinase-mediated Cassette exchange for ...
Xu Feng - One of the best experts on this subject based on the ideXlab platform.
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Development and validation of vectors containing multiple siRNA Expression Cassettes for maximizing the efficiency of gene silencing.
BMC biotechnology, 2006Co-Authors: Shunqing Wang, Zhenqi Shi, Wei Liu, Joel Jules, Xu FengAbstract:Background RNA interference (RNAi) was originally identified as a biological process in which short double-stranded RNA (dsRNA) suppress the Expression of genes complimentary to the dsRNA. This cellular intrinsic gene silencing mechanism has subsequently been developed as a useful tool for studies of gene function. A major strategy for producing small interfering RNA (siRNA) in cultured cells involves the use of siRNA Expression vectors in which a RNA polymerase III (Pol III) promoter and transcription stop signal are designed to constitute a functional siRNA Expression Cassette for production of siRNA. However, most of the available vectors contain only one siRNA Expression Cassette.
