scholarly journals Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Kalpana Dulal ◽  
Benjamin Silver ◽  
Hua Zhu
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Bac Clone ◽  
Dna Viruses ◽  
E Coli ◽  
Recombination System ◽  
Capture Method

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

scholarly journals Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi

2008 ◽  
Vol 82 (10) ◽  
pp. 4955-4964 ◽  
Author(s):  
B. Costes ◽  
G. Fournier ◽  
B. Michel ◽  
C. Delforge ◽  
V. Stalin Raj ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Thymidine Kinase ◽  
Artificial Chromosome ◽  
Wild Type ◽  
Bac Clone ◽  
Recombinant Viruses ◽  
Koi Herpesvirus ◽  
Cyprinus Carpio Koi

ABSTRACT Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

scholarly journals Bacterial Artificial Chromosome Mutagenesis Using Recombineering

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Kumaran Narayanan ◽  
Qingwen Chen
Keyword(s):  
Gene Expression ◽  
Bacterial Artificial Chromosome ◽  
Restriction Enzymes ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
E Coli ◽  
Functional Studies ◽  
Large Size

Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development ofin vivohomologous recombination strategies based on recombineering inE. colihas helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACsin vitroandin vivo.

scholarly journals Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

2001 ◽  
Vol 75 (4) ◽  
pp. 1870-1878 ◽  
Author(s):  
Andrew Marchini ◽  
Haiqing Liu ◽  
Hua Zhu
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Initial Step ◽  
Artificial Chromosome ◽  
In Vitro Assays ◽  
Bac Clone ◽  
Reading Frame ◽  
Early Genes ◽  
Direct Demonstration

ABSTRACT Much evidence suggests that the major immediate-early (IE) transactivator of human cytomegalovirus (HCMV), IE-2, is likely to be critical for efficient viral replication; however, the lack of an IE-2 mutant HCMV has precluded an experimental test of this hypothesis. As an initial step toward characterizing an IE-2 mutant, we first cloned the HCMV Towne genome as a bacterial artificial chromosome (BAC) and analyzed the ability of transfected Towne-BAC DNA (T-BACwt) to produce plaques following introduction into permissive human fibroblasts. Like Towne viral DNA, transfected T-BACwt DNA was infectious in permissive cells, and the resulting virus stocks were indistinguishable from Towne virus. We then used homologous recombination in Escherichia coli to delete the majority of UL122, the open reading frame encoding the unique portion of IE-2, from T-BACwt. From this deleted BAC, a third BAC clone in which the deletion was repaired with wild-type UL122 was created. In numerous transfections of permissive human foreskin fibroblast cells with these three BAC DNA clones, the rescued BAC and T-BACwt consistently yielded plaques, while the UL122 mutant BAC never generated plaques, even after 4 weeks. Protein and mRNA of other IE genes were readily detected from transfected UL122 mutant BAC DNA; however, reverse transcription-PCR failed to detect mRNA expression from any of five early genes examined. The generalized failure of this mutant to express early genes is consistent with expectations from in vitro assays which have demonstrated that IE-2 transactivates most HCMV promoters. These experiments provide the first direct demonstration that IE-2 is required for successful HCMV infection and indicate that virus lacking IE-2 arrests early in the replication cycle.

scholarly journals Autoexcision of Bacterial Artificial Chromosome Facilitated by Terminal Repeat-Mediated Homologous Recombination: a Novel Approach for Generating Traceless Genetic Mutants of Herpesviruses

2010 ◽  
Vol 84 (6) ◽  
pp. 2871-2880 ◽  
Author(s):  
Fuchun Zhou ◽  
Qiuhua Li ◽  
Scott W. Wong ◽  
Shou-Jiang Gao
Keyword(s):  
Homologous Recombination ◽  
Genetic Manipulation ◽  
Artificial Chromosome ◽  
Loxp Site ◽  
Terminal Repeat ◽  
Transcription Activator ◽  
Phenotypic Analysis ◽  
Bac Clone ◽  
Artificial Chromosomes

ABSTRACT Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe the use of viral intrinsic repeat sequences, which are commonly present in herpesviral genomes, to excise the BAC vector cassette. Using a newly developed in vitro transposon-based cloning approach, we obtained an infectious BAC of rhesus rhadinovirus (RRV) strain RRV26-95 with the BAC vector cassette inserted in the terminal repeat (TR) region. We showed that the BAC vector cassette was rapidly excised upon reconstitution in cells predominantly through TR-mediated homologous recombination. Genetic and phenotypic analysis showed that the BAC-excised virus was reversed to wild-type RRV. Using this autoexcisable BAC clone, we successfully generated an RRV mutant with a deletion of Orf50, which encodes a replication and transcription activator (RTA) protein. Together, these results illustrate the usefulness of TR for genetic manipulation of herpesviruses when combined with the novel transposon-based cloning approach.

scholarly journals Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8320-8329 ◽  
Author(s):  
Eva-Maria Borst ◽  
Gabriele Hahn ◽  
Ulrich H. Koszinowski ◽  
Martin Messerle
Keyword(s):  
Escherichia Coli ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Reading Frame ◽  
E Coli ◽  
Artificial Chromosomes ◽  
Internal Repeat ◽  
The Stability

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

scholarly journals A new targeted capture method using bacterial artificial chromosome (BAC) libraries as baits for sequencing relatively large genes

2018 ◽  
Author(s):  
Kae Koganebuchi ◽  
Takashi Gakuhari ◽  
Hirohiko Takeshima ◽  
Kimitoshi Sato ◽  
Kiyotaka Fujii ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Data Quality ◽  
Susceptibility Gene ◽  
Artificial Chromosome ◽  
Individual Genome ◽  
Genome Region ◽  
Capture Method ◽  
Bac Libraries ◽  
Targeted Capture ◽  
Commercial Kit

AbstractTo analyze a specific genome region using next-generation sequencing technologies, the enrichment of DNA libraries with targeted capture methods has been standardized. For enrichment of mitochondrial genome, a previous study developed an original targeted capture method that use baits constructed from long-range polymerase chain reaction (PCR) amplicons, common laboratory reagents, and equipment. In this study, a new targeted capture method is presented, that of bacterial artificial chromosome (BAC) double capture (BDC), modifying the previous method, but using BAC libraries as baits for sequencing a relatively large gene. We applied the BDC approach for the 214 kb autosomal region, ring finger protein 213, which is the susceptibility gene of moyamoya disease (MMD). To evaluate the reliability of BDC, cost and data quality were compared with those of a commercial kit. While the ratio of duplicate reads was higher, the cost was less than that of the commercial kit. The data quality was sufficiently the same as that of the kit. Thus, BDC can be an easy, low-cost, and useful method for analyzing individual genome region with substantial length.

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