The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Huanchun Chen - One of the best experts on this subject based on the ideXlab platform.
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Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel Shuttle Vector.
Veterinary microbiology, 2011Co-Authors: Liping Chen, Xuwang Cai, Fengjuan Guo, P J Blackall, Huanchun ChenAbstract:The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glässer's disease in pigs, using a novel H. parasuis-Escherichia coli Shuttle Vector. A 4.2kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning Vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a Shuttle Vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5×10(2)CFU/μg of DNA. Transformation efficiency was notably increased (1.3×10(5)CFU/μg of DNA) with Vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel Shuttle Vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen.
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Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel Shuttle Vector
Veterinary Microbiology, 2011Co-Authors: Liping Chen, Xuwang Cai, Fengjuan Guo, P J Blackall, Xu Xiaojuan, Huanchun ChenAbstract:The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glasser's disease in pigs, using a novel H. parasuis-Escherichia coli Shuttle Vector. A 4.2 kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800 bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning Vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a Shuttle Vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5 x 10(2) CFUhlg of DNA. Transformation efficiency was notably increased (1.3 x 10(5) CFU/mu g of DNA) with Vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel Shuttle Vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen. (C) 2011 Elsevier B.V. All rights reserved.
Liping Chen - One of the best experts on this subject based on the ideXlab platform.
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Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel Shuttle Vector.
Veterinary microbiology, 2011Co-Authors: Liping Chen, Xuwang Cai, Fengjuan Guo, P J Blackall, Huanchun ChenAbstract:The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glässer's disease in pigs, using a novel H. parasuis-Escherichia coli Shuttle Vector. A 4.2kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning Vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a Shuttle Vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5×10(2)CFU/μg of DNA. Transformation efficiency was notably increased (1.3×10(5)CFU/μg of DNA) with Vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel Shuttle Vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen.
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Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel Shuttle Vector
Veterinary Microbiology, 2011Co-Authors: Liping Chen, Xuwang Cai, Fengjuan Guo, P J Blackall, Xu Xiaojuan, Huanchun ChenAbstract:The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glasser's disease in pigs, using a novel H. parasuis-Escherichia coli Shuttle Vector. A 4.2 kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800 bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning Vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a Shuttle Vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5 x 10(2) CFUhlg of DNA. Transformation efficiency was notably increased (1.3 x 10(5) CFU/mu g of DNA) with Vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel Shuttle Vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen. (C) 2011 Elsevier B.V. All rights reserved.
Shiaoching Gong - One of the best experts on this subject based on the ideXlab platform.
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one step bacterial artificial chromosome bac modification cloning of the a homology arm into reporter Shuttle Vector
CSH Protocols, 2020Co-Authors: Nathaniel Heintz, Shiaoching GongAbstract:In this protocol, the homology arm sequence for one-step bacterial artificial chromosome (BAC) modification is introduced by ligation into the Shuttle Vector carrying the reporter sequence to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant Shuttle Vector. To provide further assurance that the homology box has been successfully integrated into the plasmid, the enzyme digestion pattern of the modified plasmid is compared with that of the unmodified plasmid.
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two step bacterial artificial chromosome bac engineering preparation of Shuttle Vector dna
CSH Protocols, 2020Co-Authors: Nathaniel Heintz, Shiaoching GongAbstract:In two-step bacterial artificial chromosome (BAC) engineering, a single plasmid is introduced into the BAC-carrying cell lines. The Shuttle Vector pLD53.SCAB (or pLD53.SCAEB) carries the recA gene and the R6Kγ origin, which requires the π protein to replicate. PIR2 cells, expressing π, are typically used for the amplification of the Vector and maintain about 15 copies/cell of the donor Vector, which is relatively stable in this host.
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two step bacterial artificial chromosome bac engineering electroporation of competent bac host cells with the recombinant Shuttle Vector
CSH Protocols, 2020Co-Authors: Nathaniel Heintz, Shiaoching GongAbstract:Bacterial artificial chromosome (BAC) clones are rendered electrocompetent and transformed with the recombinant Shuttle Vector, pLD53SCAB/AB-box. Cointegrates are selected by growth on chloramphenicol and ampicillin to ensure recombination of the Shuttle Vector into the BAC.
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two step bacterial artificial chromosome bac engineering preparation and verification of the recombinant Shuttle Vector
CSH Protocols, 2020Co-Authors: Nathaniel Heintz, Shiaoching GongAbstract:Plasmid DNA is prepared from the recombinant Shuttle Vector pLD53.SCAB/A-B created by cloning of the A and B homology arms for two-step bacterial artificial chromosome (BAC) engineering. To confirm that the A-box and B-box arms have been successfully incorporated into pLD53.SCAB, the pattern of enzyme digestion of the modified plasmid is compared with that of the unmodified pLD53.SCAB. Once the Shuttle Vector is shown to carry the proper sequences, it is ready for transfer into the BAC host.
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two step bacterial artificial chromosome bac engineering cloning of the a and b homology arms into the Shuttle Vector
CSH Protocols, 2020Co-Authors: Nathaniel Heintz, Shiaoching GongAbstract:This protocol describes the preparation of the Shuttle Vector before its introduction into bacterial artificial chromosome (BAC) host cells for BAC two-step engineering. The homology arm sequences, prepared previously, are introduced by ligation into the digested Shuttle Vector DNA to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant Shuttle Vector.
Faustino Siñeriz - One of the best experts on this subject based on the ideXlab platform.
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Construction of an integrative Shuttle Vector for Zymomonas mobilis
FEMS Microbiology Letters, 1995Co-Authors: Osvaldo D. Delgado, Carlos Mauricio Abate, Faustino SiñerizAbstract:An integrative Shuttle Vector, pZMOCPI, was constructed by ligating EcoRV digests of the plasmid cloning Vector pBluescript and pZMPI, a cryptic plasmid of Zymomonas mobilis PROM Al. The 7.2-kb plasmid pZMOCPI replicated in Escherichia coli and could also be transferred from this host by electroporation to Z. mobilis ATCC 29191. The transformants were selected by ampicillin resistance. The integrative characteristic was detected by hybridization in situ. The Vector was stably maintained in Z. mobilis after 200 generations without selective pressure. © 1995.
Xilong Xiao - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of mutagenesis induced by olaquindox using a Shuttle Vector psp189 mammalian cell system
Mutation Research, 2006Co-Authors: Lihua Hao, Qian Chen, Xilong XiaoAbstract:Olaquindox, a quinoxaline 1,4-dioxide derivative from quindoxin, is widely used as an animal growth promoter in China. We tested olaquindox as a mutagen in a SV40-based Shuttle Vector pSP189 and African green kidney cell (Vero E6 cell line) system to define the safety of olaquindox as a food-additive for animals. When applied at 6.6 microg/ml, olaquindox caused 12 times higher mutation frequency in comparison to untreated controls. More than 70% of base substitutions happened at G:C base pairs featuring G:C to T:A or G:C to A:T conversions. Frequency of point mutations for in vitro modified plasmids was also dramatically increased from the spontaneous background level. Olaquindox-induced mutations did not occur randomly along the supF Shuttle Vector, but instead, had a hot spot at base pair #155 which accounts for 37% of total mutations. Olaquindox-induced mutations also showed sequence-specificity in which most point mutations occurred at site N in a 5'-NNTTNN-3' sequence while most tandem bases deletion and rearrangement were seen at the 5'-ANGGCCNAAA-3' sequence. We conclude that olaquindox induces DNA mutation, therefore, should not be used as an additive to promote animal growth.
