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Shiaoching Gong - One of the best experts on this subject based on the ideXlab platform.

  • one step Bacterial Artificial Chromosome bac modification preparation of plasmids
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    In the one-step approach to Bacterial Artificial Chromosome (BAC) modification, two plasmids are introduced into the BAC host cells. The shuttle pLD53.SC2, carrying the EFGP reporter sequence and requiring the π protein to replicate, must be grown in PIR1- or PIR2-competent Escherichia coli Our preference for these vectors is PIR1, because these cells are able to maintain about 250 copies of the donor vector. This small-sized vector is stable in PIR1. The RecA plasmid pSV1.RecA has a temperature-sensitive origin of replication and can be grown in most competent bacteria at 30°C; here we use DH5α competent cells. This protocol describes preparation of the vector DNAs. The shuttle-reporter vector DNA is subsequently digested for introduction of one homology arm (typically the A-box).

  • one step Bacterial Artificial Chromosome bac modification transformation of the bac host with the reca vector
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    This protocol outlines the steps for introducing the RecA plasmid into Bacterial Artificial Chromosome (BAC) host cells, and their preparation for subsequent transformation with the reporter plasmid for one-step BAC modification. BAC host cells are rendered chemically competent and transformed with the RecA plasmid, and transformants are selected for tetracycline resistance to ensure the presence of the RecA marker.

  • one step Bacterial Artificial Chromosome bac modification preparation of the a homology arm a box
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    The one-step approach to Bacterial Artificial Chromosome (BAC) modification requires that only one homology arm be cloned into the shuttle vector (in the example presented here, we use the "A-box"). The homology arm, which in this case lies upstream of the ATG start codon, is amplified by polymerase chain reaction (PCR) using purified BAC DNA as template. The resulting amplification product is then digested with the appropriate restriction endonuclease to render it suitable for cloning into the shuttle vector.

  • one step Bacterial Artificial Chromosome bac modification cloning of the a homology arm into reporter shuttle vector
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    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.

  • one step Bacterial Artificial Chromosome bac modification transfer of the reporter vector into bac reca cells and selection of co integrates
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    In one-step Bacterial Artificial Chromosome (BAC) modification, recombination of the reporter vector with the BAC-leading to the modified BAC-is facilitated by the presence of RecA. Recombinants are selected for by growth in the presence of chloramphenicol, ampicillin, and tetracycline. Only bacteria containing correctly modified BACs and copies of pSV1.RecA will be selected. Unmodified BACs (i.e., those lacking a pLD53.SC2/A-box insert) are eliminated by exposure to ampicillin. Free reporter plasmid remaining in the BAC host bacteria will also be eliminated, because this vector requires the π protein to replicate. The co-integrates are selected by growth at high temperature, thereby eliminating the RecA plasmid. Successful modification of the BAC-formation of the co-integrate-is confirmed in separate amplification reactions.

Nathaniel Heintz - One of the best experts on this subject based on the ideXlab platform.

  • one step Bacterial Artificial Chromosome bac modification preparation of plasmids
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    In the one-step approach to Bacterial Artificial Chromosome (BAC) modification, two plasmids are introduced into the BAC host cells. The shuttle pLD53.SC2, carrying the EFGP reporter sequence and requiring the π protein to replicate, must be grown in PIR1- or PIR2-competent Escherichia coli Our preference for these vectors is PIR1, because these cells are able to maintain about 250 copies of the donor vector. This small-sized vector is stable in PIR1. The RecA plasmid pSV1.RecA has a temperature-sensitive origin of replication and can be grown in most competent bacteria at 30°C; here we use DH5α competent cells. This protocol describes preparation of the vector DNAs. The shuttle-reporter vector DNA is subsequently digested for introduction of one homology arm (typically the A-box).

  • one step Bacterial Artificial Chromosome bac modification transformation of the bac host with the reca vector
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    This protocol outlines the steps for introducing the RecA plasmid into Bacterial Artificial Chromosome (BAC) host cells, and their preparation for subsequent transformation with the reporter plasmid for one-step BAC modification. BAC host cells are rendered chemically competent and transformed with the RecA plasmid, and transformants are selected for tetracycline resistance to ensure the presence of the RecA marker.

  • one step Bacterial Artificial Chromosome bac modification preparation of the a homology arm a box
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    The one-step approach to Bacterial Artificial Chromosome (BAC) modification requires that only one homology arm be cloned into the shuttle vector (in the example presented here, we use the "A-box"). The homology arm, which in this case lies upstream of the ATG start codon, is amplified by polymerase chain reaction (PCR) using purified BAC DNA as template. The resulting amplification product is then digested with the appropriate restriction endonuclease to render it suitable for cloning into the shuttle vector.

  • one step Bacterial Artificial Chromosome bac modification cloning of the a homology arm into reporter shuttle vector
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    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.

  • one step Bacterial Artificial Chromosome bac modification transfer of the reporter vector into bac reca cells and selection of co integrates
    CSH Protocols, 2020
    Co-Authors: Nathaniel Heintz, Shiaoching Gong
    Abstract:

    In one-step Bacterial Artificial Chromosome (BAC) modification, recombination of the reporter vector with the BAC-leading to the modified BAC-is facilitated by the presence of RecA. Recombinants are selected for by growth in the presence of chloramphenicol, ampicillin, and tetracycline. Only bacteria containing correctly modified BACs and copies of pSV1.RecA will be selected. Unmodified BACs (i.e., those lacking a pLD53.SC2/A-box insert) are eliminated by exposure to ampicillin. Free reporter plasmid remaining in the BAC host bacteria will also be eliminated, because this vector requires the π protein to replicate. The co-integrates are selected by growth at high temperature, thereby eliminating the RecA plasmid. Successful modification of the BAC-formation of the co-integrate-is confirmed in separate amplification reactions.

Andrew K. Groves - One of the best experts on this subject based on the ideXlab platform.

  • generation of pax2 cre mice by modification of a pax2 Bacterial Artificial Chromosome
    Genesis, 2004
    Co-Authors: Takahiro Ohyama, Andrew K. Groves
    Abstract:

    Summary: The Pax2 gene is expressed in the developing otocyst, kidney, and midbrain–hindbrain boundary. We generated Pax2-Cre transgenic lines by modification of a Pax2 Bacterial Artificial Chromosome (BAC). In one Pax2-Cre line, Cre mRNA starts to be expressed in the otic placode at the late presomite stage. R26R reporter mouse analysis revealed that the Cre expression is sufficient to delete the loxP-flanked sequences in most of the cells in the inner ear. Reporter-positive cells are also detected in other Pax2-expressing tissues such as midbrain, cerebellum, olfactory bulb, and kidney, suggesting that these cells are the descendants of Pax2-expressing cells in these tissues and that Pax2-Cre transgenic mice can delete genes efficiently in these tissues. genesis 38:195–199, 2004. © 2004 Wiley-Liss, Inc.

  • generation of pax2 cre mice by modification of a pax2 Bacterial Artificial Chromosome
    Genesis, 2004
    Co-Authors: Takahiro Ohyama, Andrew K. Groves
    Abstract:

    The Pax2 gene is expressed in the developing otocyst, kidney, and midbrain-hindbrain boundary. We generated Pax2-Cre transgenic lines by modification of a Pax2 Bacterial Artificial Chromosome (BAC). In one Pax2-Cre line, Cre mRNA starts to be expressed in the otic placode at the late presomite stage. R26R reporter mouse analysis revealed that the Cre expression is sufficient to delete the loxP-flanked sequences in most of the cells in the inner ear. Reporter-positive cells are also detected in other Pax2-expressing tissues such as midbrain, cerebellum, olfactory bulb, and kidney, suggesting that these cells are the descendants of Pax2-expressing cells in these tissues and that Pax2-Cre transgenic mice can delete genes efficiently in these tissues.

Rod A. Wing - One of the best experts on this subject based on the ideXlab platform.

  • construction of plant Bacterial Artificial Chromosome bac libraries an illustrated guide
    Journal of Agricultural Genomics, 2000
    Co-Authors: Jeffrey Tomkins, Rod A. Wing, David Frisch, Andrew H Paterson
    Abstract:

    J. Agric. Genomics, 5 ABSTRACT Bacterial Artificial Chromosome (BAC) libraries have become invaluable tools in plant genetic research. However, it is difficult for new practitioners to create plant BAC libraries de novo because published protocols are not particularly detailed, and plant cells possess features that make isolation of clean, high molecular weight DNA troublesome. In this document we present an illustrated, step-by-step protocol for constructing plant BAC libraries. This protocol is sufficiently detailed to be of use to both new and experienced investigators. We hope that by reducing the obstacles to BAC cloning in plants, we will foster new and accelerated progress in plant genomics.

  • construction and characterization of a Bacterial Artificial Chromosome bac library for the a genome of wheat
    Genome, 1999
    Co-Authors: Diego Lijavetzky, Rod A. Wing, G Muzzi, Thomas Wicker, Beat Keller, Jorge Dubcovsky
    Abstract:

    A genomic Bacterial Artificial Chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276,480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720,384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for Chromosome walking.

  • a Bacterial Artificial Chromosome library for sugarcane
    Theoretical and Applied Genetics, 1999
    Co-Authors: Jeffrey Tomkins, David Frisch, Sung Sick Woo, H Millersmith, Rod A. Wing
    Abstract:

    Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits in sugarcane show complex inheritance, a rust locus showing monogenic inheritance has been documented. In order to facilitate cloning of the rust locus, we have constructed a Bacterial Artificial Chromosome (BAC) library for the cultivar R570. The library contains 103,296 clones providing 4.5 sugarcane genome equivalents. A random sampling of 240 clones indicated an average insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm2 filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of less than 1%. To demonstrate the library’s potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted, and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene.

  • construction and characterization of a bovine Bacterial Artificial Chromosome library
    Genomics, 1995
    Co-Authors: Li I Cai, Rod A. Wing, J F Taylor, D S Gallagher, Sung Sick Woo, Scott K Davis
    Abstract:

    A Bacterial Artificial Chromosome (BAC) library has been constructed for use in bovine genome mapping using the pBeloBAC11 vector. Currently, the library consists of 23,040 clones, which achieves a 70% probability (P= 0.70) of the library containing a specific unique DNA sequence. Sixty thousand clones, or about three haploid bovine genomes, will be required to achieve a 95% probability (P= 0.95) of containing a unique sequence. An average insert size of 146 kb was estimated from the analysis of 77 randomly selected BAC clones produced by one or two rounds of size selection. The bovine DNA inserts proved to be very stable for at least 100 cell generations. No chimeric clones were detected among 11 large, size-selected BAC clones using fluorescencein situhybridization (FISH) on metaphase bovine Chromosomes. The polymerase chain reaction (PCR) was used to screen the library for single-copy nuclear sequences. Thirty-three of 46 (72%) sequences were present in the library in at least one copy, which is consistent with the estimated 70% probability of this library containing a unique DNA sequence. A BAC clone containing the 3β-hydroxy-5-ene steroid dehydrogenase (HSD3B) gene was physically mapped to bovine Chromosome 3 by FISH. Two new microsatellite markers were isolated from the HSD3B-positive BAC clone as sequence-tagged sites for genetic mapping. These markers cosegregated, and no recombinants were detected in 193 informative meioses. Plasmid end rescue and the inverse polymerase chain reaction methods were used to rescue both ends of this BAC clone, and Chromosome walking was performed using PCR primers designed within the end region sequences. Based on our experimental results, the BAC system provides a very useful tool for complex genome analysis.

  • construction and characterization of a bovine Bacterial Artificial Chromosome library
    Genomics, 1995
    Co-Authors: Li Cai, Rod A. Wing, J F Taylor, D S Gallagher, Sung Sick Woo, Scott K Davis
    Abstract:

    A Bacterial Artificial Chromosome (BAC) library has been constructed for use in bovine genome mapping using constructed for use in bovine genome mapping using the pBeloBAC11 vector. Currently, the library consists of 23,040 clones, which achieves a 70% probability (P=0.70) of the library containing a specific unique DNA sequence. Sixty thousand clones, or about three haploid bovine genomes, will be required to achieve a 95% probability (P=0.95) of containing a unique sequence. An average insert size of 146 kb was estimated from the analysis of 77 randomly selected BAC clones produced by one or two rounds of size selection. The bovine DNA inserts proved to be very stable for at least 100 cell generations. No chimeric clones were detected among 11 large, size-selected BAC clones using fluorescence in situ hybridization (FISH) on metaphase bovine Chromosomes. Thirty-three of 46 (72%) sequences were present in the library in at least one copy, which is consistent with the estimated 70% probability of this library containing a unique DNA sequence. A BAC clone as sequence-tagged sites for genetic mapping. These markers cosegregated, and no recombinants were detected in 193 informative meioses. Plasmid end rescue and the inverse polymerase chain reaction methods were used to rescue both ends of this BAC clone, and Chromosome walking was performed using PCR primers designed within the end region sequences. Based on our experimental results, the BAC system provides a very useful tool for complex genome analysis.

Ulrich H Koszinowski - One of the best experts on this subject based on the ideXlab platform.

  • development of bovine herpesvirus 4 as an expression vector using Bacterial Artificial Chromosome cloning
    Journal of General Virology, 2005
    Co-Authors: Laurent Gillet, Nicolas Markinegoriaynoff, Virginie Daix, Gaetano Donofrio, Ulrich H Koszinowski, Markus Wagner, B China, Mathias Ackermann, Alain Vanderplasschen
    Abstract:

    Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting Bacterial Artificial Chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.

  • cloning of the varicella zoster virus genome as an infectious Bacterial Artificial Chromosome in escherichia coli
    Vaccine, 2004
    Co-Authors: Kazuhiro Nagaike, Ulrich H Koszinowski, Markus Wagner, Hironori Yoshii, Michiaki Takahashi, Yasuko Mori, Yasuyuki Gomi, Koichi Yamanishi
    Abstract:

    The complete genome of the varicella-zoster virus (VZV) Oka strain has been cloned as a Bacterial Artificial Chromosome (BAC). Following electroporation into Escherichia coli (E. coli) strain DH10B, the VZV BAC was stably propagated over multiple generations of its host. Human embryonic lung (HEL) cells transfected with VZV BAC DNA recovered from DH10B showed cytopathic effect (CPE), and virus spread to neighbouring cells was observed. BAC vector sequences are flanked by loxP sites and, coinfection of the reconstituted virus, with a recombinant adenovirus expressing Cre recombinase removed the Bacterial sequences. The resulting recombinant rV02 grew as well as the parental virus in HEL cells. The recombinant VZV will promote VZV research and increase use of the viral genome as an investigative tool.

  • cloning and mutagenesis of a herpesvirus genome as an infectious Bacterial Artificial Chromosome
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Martin Messerle, Irena Crnkovic, Wolfgang Hammerschmidt, Heike Ziegler, Ulrich H Koszinowski
    Abstract:

    A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb Bacterial Artificial Chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.