Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones
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 inside E. coli allows for accurate recombinant virus generation....
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doaj-5b9155c1f7164e02b20409624d609b422020-11-25T02:03:38ZengHindawi LimitedJournal of Biomedicine and Biotechnology1110-72431110-72512012-01-01201210.1155/2012/357147357147Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome ClonesKalpana Dulal0Benjamin Silver1Hua Zhu2Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, 225 Warren Street, Newark, New Jersey 07101-1709, USADepartment of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, 225 Warren Street, Newark, New Jersey 07101-1709, USADepartment of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, 225 Warren Street, Newark, New Jersey 07101-1709, USABacterial 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 inside E. coli allows 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 in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.http://dx.doi.org/10.1155/2012/357147 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kalpana Dulal Benjamin Silver Hua Zhu |
spellingShingle |
Kalpana Dulal Benjamin Silver Hua Zhu Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones Journal of Biomedicine and Biotechnology |
author_facet |
Kalpana Dulal Benjamin Silver Hua Zhu |
author_sort |
Kalpana Dulal |
title |
Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones |
title_short |
Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones |
title_full |
Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones |
title_fullStr |
Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones |
title_full_unstemmed |
Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones |
title_sort |
use of recombination-mediated genetic engineering for construction of rescue human cytomegalovirus bacterial artificial chromosome clones |
publisher |
Hindawi Limited |
series |
Journal of Biomedicine and Biotechnology |
issn |
1110-7243 1110-7251 |
publishDate |
2012-01-01 |
description |
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 inside E. coli allows 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 in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning. |
url |
http://dx.doi.org/10.1155/2012/357147 |
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1724946832945053696 |