The Experts below are selected from a list of 90 Experts worldwide ranked by ideXlab platform
David S Roos - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of differentiation mutants in the protozoan parasite toxoplasma gondii
Molecular Microbiology, 2002Co-Authors: Mariana Matrajt, Robert G K Donald, Upinder Singh, David S RoosAbstract:Summary Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphori- bosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by brady- zoite induction in vitro and selection in 6-Thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachy- zoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under brady- zoite differentiation conditions.
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gene knock outs and allelic replacements in toxoplasma gondii hxgprt as a selectable marker for hit and run mutagenesis
Molecular and Biochemical Parasitology, 1998Co-Authors: Robert G K Donald, David S RoosAbstract:Abstract The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene of the protozoan parasite Toxoplasma gondii encodes a safe, practical genetic marker suitable for both positive and negative selection. Taking advantage of the ability to control homologous versus nonhomologous recombination in haploid T. gondii tachyzoites by manipulating the length of homologous DNA sequence, we have explored the possibility of ‘hit-and-run’ mutagenesis to introduce gene knock-outs (or allelic replacements) at loci for which no known selection or screen is available. Using the uracil phosphoribosyl transferase (UPRT) locus as a target, a genomic clone containing ≈8 kb encompassing the UPRT gene (but lacking essential coding sequence) was fused to a cDNA-derived HXGPRT ‘minigene’, which lacks sufficient continguous genomic sequence for homologous recombination. After transfection of circular plasmid DNA, positive selection for HXGPRT activity identified stable transformants, >30% of which were found to have integrated at the UPRT locus as ‘pseudodiploids’ (produced by single-site homologous recombination between the circular plasmid and genomic DNA). Upon removal of mycophenolic acid, resolution of pseudodiploids by spontaneous intrachromosomal homologous recombination was selected using 6-Thioxanthine, yielding a 1:1 ratio of UPRT knock-out parasites to wild-type revertants, at frequencies of ≈10 –6 per parasite doubling. Applications of ‘hit-and-run’ technology relative to other gene targeting strategies are discussed.
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insertional tagging cloning and expression of the toxoplasma gondii hypoxanthine xanthine guanine phosphoribosyltransferase gene use as a selectable marker for stable transformation
Journal of Biological Chemistry, 1996Co-Authors: Robert G K Donald, Darrick Carter, Buddy Ullman, David S RoosAbstract:Abstract A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-Thioxanthine arose at a frequency of ∼3 × 10−7. Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create “knock-out” parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.
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insertional tagging cloning and expression of the toxoplasma gondii hypoxanthine xanthine guanine phosphoribosyltransferase gene use as a selectable marker for stable transformation
Journal of Biological Chemistry, 1996Co-Authors: Robert G K Donald, Darrick Carter, Buddy Ullman, David S RoosAbstract:A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-Thioxanthine arose at a frequency of approximately3 x 10(-7). Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create "knock-out" parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.
Antonio E Chiocca - One of the best experts on this subject based on the ideXlab platform.
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regression of experimental brain tumors with 6 Thioxanthine and escherichia coli gpt gene therapy
Human Gene Therapy, 1997Co-Authors: Yasuhiro Ono, Keiro Ikeda, Ming X Wei, Griffith R Harsh, Takashi Tamiya, Antonio E ChioccaAbstract:ABSTRACT The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-Thioxanthine, with an ID50 of 4.1 μM, whereas parental C6 cells were not affected at a concentration of 500 μM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This “modified” bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retro...
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regression of experimental brain tumors with 6 Thioxanthine and escherichia coli gpt gene therapy
Human Gene Therapy, 1997Co-Authors: Yasuhiro Ono, Keiro Ikeda, Ming X Wei, Griffith R Harsh, Takashi Tamiya, Antonio E ChioccaAbstract:ABSTRACT The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6...
Yasuhiro Ono - One of the best experts on this subject based on the ideXlab platform.
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regression of experimental brain tumors with 6 Thioxanthine and escherichia coli gpt gene therapy
Human Gene Therapy, 1997Co-Authors: Yasuhiro Ono, Keiro Ikeda, Ming X Wei, Griffith R Harsh, Takashi Tamiya, Antonio E ChioccaAbstract:ABSTRACT The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-Thioxanthine, with an ID50 of 4.1 μM, whereas parental C6 cells were not affected at a concentration of 500 μM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This “modified” bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retro...
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regression of experimental brain tumors with 6 Thioxanthine and escherichia coli gpt gene therapy
Human Gene Therapy, 1997Co-Authors: Yasuhiro Ono, Keiro Ikeda, Ming X Wei, Griffith R Harsh, Takashi Tamiya, Antonio E ChioccaAbstract:ABSTRACT The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6...
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escherichia coli gpt gene sensitizes rat glioma cells to killing by 6 Thioxanthine or 6 thioguanine
Cancer Gene Therapy, 1996Co-Authors: Takashi Tamiya, Yasuhiro Ono, Ming X Wei, P J Mroz, F L Moolte, E A ChioccaAbstract:: Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-Thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.
Takashi Tamiya - One of the best experts on this subject based on the ideXlab platform.
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regression of experimental brain tumors with 6 Thioxanthine and escherichia coli gpt gene therapy
Human Gene Therapy, 1997Co-Authors: Yasuhiro Ono, Keiro Ikeda, Ming X Wei, Griffith R Harsh, Takashi Tamiya, Antonio E ChioccaAbstract:ABSTRACT The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-Thioxanthine, with an ID50 of 4.1 μM, whereas parental C6 cells were not affected at a concentration of 500 μM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This “modified” bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retro...
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regression of experimental brain tumors with 6 Thioxanthine and escherichia coli gpt gene therapy
Human Gene Therapy, 1997Co-Authors: Yasuhiro Ono, Keiro Ikeda, Ming X Wei, Griffith R Harsh, Takashi Tamiya, Antonio E ChioccaAbstract:ABSTRACT The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6...
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escherichia coli gpt gene sensitizes rat glioma cells to killing by 6 Thioxanthine or 6 thioguanine
Cancer Gene Therapy, 1996Co-Authors: Takashi Tamiya, Yasuhiro Ono, Ming X Wei, P J Mroz, F L Moolte, E A ChioccaAbstract:: Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-Thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.
Robert G K Donald - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of differentiation mutants in the protozoan parasite toxoplasma gondii
Molecular Microbiology, 2002Co-Authors: Mariana Matrajt, Robert G K Donald, Upinder Singh, David S RoosAbstract:Summary Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphori- bosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by brady- zoite induction in vitro and selection in 6-Thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachy- zoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under brady- zoite differentiation conditions.
-
gene knock outs and allelic replacements in toxoplasma gondii hxgprt as a selectable marker for hit and run mutagenesis
Molecular and Biochemical Parasitology, 1998Co-Authors: Robert G K Donald, David S RoosAbstract:Abstract The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene of the protozoan parasite Toxoplasma gondii encodes a safe, practical genetic marker suitable for both positive and negative selection. Taking advantage of the ability to control homologous versus nonhomologous recombination in haploid T. gondii tachyzoites by manipulating the length of homologous DNA sequence, we have explored the possibility of ‘hit-and-run’ mutagenesis to introduce gene knock-outs (or allelic replacements) at loci for which no known selection or screen is available. Using the uracil phosphoribosyl transferase (UPRT) locus as a target, a genomic clone containing ≈8 kb encompassing the UPRT gene (but lacking essential coding sequence) was fused to a cDNA-derived HXGPRT ‘minigene’, which lacks sufficient continguous genomic sequence for homologous recombination. After transfection of circular plasmid DNA, positive selection for HXGPRT activity identified stable transformants, >30% of which were found to have integrated at the UPRT locus as ‘pseudodiploids’ (produced by single-site homologous recombination between the circular plasmid and genomic DNA). Upon removal of mycophenolic acid, resolution of pseudodiploids by spontaneous intrachromosomal homologous recombination was selected using 6-Thioxanthine, yielding a 1:1 ratio of UPRT knock-out parasites to wild-type revertants, at frequencies of ≈10 –6 per parasite doubling. Applications of ‘hit-and-run’ technology relative to other gene targeting strategies are discussed.
-
insertional tagging cloning and expression of the toxoplasma gondii hypoxanthine xanthine guanine phosphoribosyltransferase gene use as a selectable marker for stable transformation
Journal of Biological Chemistry, 1996Co-Authors: Robert G K Donald, Darrick Carter, Buddy Ullman, David S RoosAbstract:Abstract A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-Thioxanthine arose at a frequency of ∼3 × 10−7. Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create “knock-out” parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.
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insertional tagging cloning and expression of the toxoplasma gondii hypoxanthine xanthine guanine phosphoribosyltransferase gene use as a selectable marker for stable transformation
Journal of Biological Chemistry, 1996Co-Authors: Robert G K Donald, Darrick Carter, Buddy Ullman, David S RoosAbstract:A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-Thioxanthine arose at a frequency of approximately3 x 10(-7). Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create "knock-out" parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.
