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A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red reco...

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Autores principales: Jain, Vaibhav, Plaisance-Bonstaff, Karlie, Sangani, Rajnikumar, Lanier, Curtis, Dolce, Alexander, Hu, Jianhong, Brulois, Kevin, Haecker, Irina, Turner, Peter, Renne, Rolf, Krueger, Brian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776209/
https://www.ncbi.nlm.nih.gov/pubmed/26907327
http://dx.doi.org/10.3390/v8020054
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author Jain, Vaibhav
Plaisance-Bonstaff, Karlie
Sangani, Rajnikumar
Lanier, Curtis
Dolce, Alexander
Hu, Jianhong
Brulois, Kevin
Haecker, Irina
Turner, Peter
Renne, Rolf
Krueger, Brian
author_facet Jain, Vaibhav
Plaisance-Bonstaff, Karlie
Sangani, Rajnikumar
Lanier, Curtis
Dolce, Alexander
Hu, Jianhong
Brulois, Kevin
Haecker, Irina
Turner, Peter
Renne, Rolf
Krueger, Brian
author_sort Jain, Vaibhav
collection PubMed
description Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community.
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spelling pubmed-47762092016-03-09 A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants Jain, Vaibhav Plaisance-Bonstaff, Karlie Sangani, Rajnikumar Lanier, Curtis Dolce, Alexander Hu, Jianhong Brulois, Kevin Haecker, Irina Turner, Peter Renne, Rolf Krueger, Brian Viruses Article Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community. MDPI 2016-02-19 /pmc/articles/PMC4776209/ /pubmed/26907327 http://dx.doi.org/10.3390/v8020054 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Jain, Vaibhav
Plaisance-Bonstaff, Karlie
Sangani, Rajnikumar
Lanier, Curtis
Dolce, Alexander
Hu, Jianhong
Brulois, Kevin
Haecker, Irina
Turner, Peter
Renne, Rolf
Krueger, Brian
A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants
title A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants
title_full A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants
title_fullStr A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants
title_full_unstemmed A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants
title_short A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants
title_sort toolbox for herpesvirus mirna research: construction of a complete set of kshv mirna deletion mutants
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776209/
https://www.ncbi.nlm.nih.gov/pubmed/26907327
http://dx.doi.org/10.3390/v8020054
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