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Michel Jadot - One of the best experts on this subject based on the ideXlab platform.
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Hydrodynamics-Based Transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection.
The journal of gene medicine, 2004Co-Authors: Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-de Coninck, Robert Wattiaux, Michel JadotAbstract:Background The mechanism of gene transfer into hepatocytes by the Hydrodynamics-Based Transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the Transfection. Methods We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. Results When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of 35S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. Conclusions Our results show that the efficiency of Hydrodynamics-Based Transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure. Copyright © 2004 John Wiley & Sons, Ltd.
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140. Hydrodynamics-Based Transfection of the liver: entrance into hepatocytes of DNA that causes expression, takes place very early after injection
Molecular Therapy, 2004Co-Authors: Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-de Coninck, Robert Wattiaux, Michel JadotAbstract:The mechanism of gene transfer into hepatocytes by the Hydrodynamics-Based Transfection procedure is not clearly understood. It has been shown that after an hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible of the efficiency of the Transfection (Lecocq M et al, J of Gene Med, 5, 142-156, 2003). We have investigated the problem by giving to the mice an hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we have determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by an hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other hand.
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Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume.
The journal of gene medicine, 2003Co-Authors: Michèle Lecocq, Fanjam Andrianaivo, Simone Wattiaux-de Coninck, Robert Wattiaux, Marie‐thérèse Warnier, Michel JadotAbstract:BACKGROUND An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (Hydrodynamics-Based Transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA. METHODS We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes). RESULTS (35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face. CONCLUSIONS The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour Transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA.
Pablo C D. Ghiringhelli - One of the best experts on this subject based on the ideXlab platform.
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Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo, Leading to Production of Biologically Active Stx2
2016Co-Authors: Luciano P. Pedrotti, Jorge D Goldstein, Pablo C D. Ghiringhelli, Marina B S. PalermoaAbstract:ABSTRACT Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We pre-viously described Stx2 expression by eukaryotic cells after they were transfected in vitrowith the stx2 gene cloned into a prokary-otic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by Hydrodynamics-Based Transfection (HBT) with pStx2. We studied the survival, percent-age of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNAwas recovered from the liver, and Stx2 cytotoxic activity was ob-served in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, andmarkers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx2 gene, reproducing pathogenic damage in
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Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo, Leading to Production of Biologically Active Stx2
mBio, 2013Co-Authors: Leticia Verónica Bentancor, Luciano P. Pedrotti, Maria Pilar Mejias, Alipio Pinto, Marcos Fabián Bilen, Roberto Meiss, Maria Cecilia Rodriguez-galan, Natalia S. Baez, Jorge Goldstein, Pablo C D. GhiringhelliAbstract:Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We pre- viously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx 2 gene cloned into a prokary- otic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by Hydrodynamics-Based Transfection (HBT) with pStx2. We studied the survival, percent- age of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was ob- served in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glialfibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx 2 gene, reproducing pathogenic damage in- duced by purified Stx2 or secondary to EHEC infection. IMPORTANCE Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the de- tailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter as- sociated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since lo- cal eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.
Y K Song - One of the best experts on this subject based on the ideXlab platform.
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Long-term expression of human alpha1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a Hydrodynamics-Based procedure.
Gene therapy, 2000Co-Authors: Guisheng Zhang, Y K Song, Dexi LiuAbstract:The liver is an important target organ for gene transfer due to its large capacity for synthesizing serum proteins and its involvement in numerous genetic and acquired diseases. Previously, we and others have shown that an efficient gene transfer to liver cells in vivo can be achieved by an intravenous injection of plasmid DNA using a Hydrodynamics-Based procedure. In this study, we systematically characterized the expression of transgene encoding a secretory protein in mouse. Using human alpha1-antitrypsin (hAAT) gene as a reporter, we demonstrate that the serum level of hAAT can reach as high as 0.5 mg/ml by a simple tail vein injection of 10-50 microg plasmid DNA into a mouse. The serum hAAT reaches the peak level 1 day after DNA injection and then declines during the following 2 to 4 weeks to 2-5 microg/ml, a level which persists for at least 6 months. Southern analysis of extracted DNA and RT-PCR analysis of RNA from the liver reveal that hAAT gene is active and present as episomal form after 6 months. These results suggest that the Hydrodynamics-Based Transfection procedure provides a valuable tool for screening genes for therapeutic purposes in whole animals.
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Hydrodynamics Based Transfection in animals by systemic administration of plasmid dna
Gene Therapy, 1999Co-Authors: Y K SongAbstract:Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a Hydrodynamics-Based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and β-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 μg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 μg of plasmid DNA into a mouse. Histochemical analysis using β-galactosidase gene as a reporter reveals that approximately 40% of hepatocytes express the transgene. The time–response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.
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Hydrodynamics-Based Transfection in animals by systemic administration of plasmid DNA
Gene Therapy, 1999Co-Authors: F Liu, Y K Song, D. LiuAbstract:Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a Hydrodynamics-Based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.
Fanjam Andrianaivo - One of the best experts on this subject based on the ideXlab platform.
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Hydrodynamics-Based Transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection.
The journal of gene medicine, 2004Co-Authors: Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-de Coninck, Robert Wattiaux, Michel JadotAbstract:Background The mechanism of gene transfer into hepatocytes by the Hydrodynamics-Based Transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the Transfection. Methods We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. Results When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of 35S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. Conclusions Our results show that the efficiency of Hydrodynamics-Based Transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure. Copyright © 2004 John Wiley & Sons, Ltd.
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140. Hydrodynamics-Based Transfection of the liver: entrance into hepatocytes of DNA that causes expression, takes place very early after injection
Molecular Therapy, 2004Co-Authors: Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-de Coninck, Robert Wattiaux, Michel JadotAbstract:The mechanism of gene transfer into hepatocytes by the Hydrodynamics-Based Transfection procedure is not clearly understood. It has been shown that after an hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible of the efficiency of the Transfection (Lecocq M et al, J of Gene Med, 5, 142-156, 2003). We have investigated the problem by giving to the mice an hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we have determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by an hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other hand.
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Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume.
The journal of gene medicine, 2003Co-Authors: Michèle Lecocq, Fanjam Andrianaivo, Simone Wattiaux-de Coninck, Robert Wattiaux, Marie‐thérèse Warnier, Michel JadotAbstract:BACKGROUND An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (Hydrodynamics-Based Transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA. METHODS We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes). RESULTS (35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face. CONCLUSIONS The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour Transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA.
Michèle Lecocq - One of the best experts on this subject based on the ideXlab platform.
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Hydrodynamics-Based Transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection.
The journal of gene medicine, 2004Co-Authors: Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-de Coninck, Robert Wattiaux, Michel JadotAbstract:Background The mechanism of gene transfer into hepatocytes by the Hydrodynamics-Based Transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the Transfection. Methods We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. Results When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of 35S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. Conclusions Our results show that the efficiency of Hydrodynamics-Based Transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure. Copyright © 2004 John Wiley & Sons, Ltd.
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140. Hydrodynamics-Based Transfection of the liver: entrance into hepatocytes of DNA that causes expression, takes place very early after injection
Molecular Therapy, 2004Co-Authors: Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-de Coninck, Robert Wattiaux, Michel JadotAbstract:The mechanism of gene transfer into hepatocytes by the Hydrodynamics-Based Transfection procedure is not clearly understood. It has been shown that after an hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible of the efficiency of the Transfection (Lecocq M et al, J of Gene Med, 5, 142-156, 2003). We have investigated the problem by giving to the mice an hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we have determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by an hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other hand.
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Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume.
The journal of gene medicine, 2003Co-Authors: Michèle Lecocq, Fanjam Andrianaivo, Simone Wattiaux-de Coninck, Robert Wattiaux, Marie‐thérèse Warnier, Michel JadotAbstract:BACKGROUND An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (Hydrodynamics-Based Transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA. METHODS We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes). RESULTS (35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face. CONCLUSIONS The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour Transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA.
