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Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes

The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in recent years sparked the development of transgenic technologies with the potential to be used as novel vector control tools. These technologies rely on genome editing that confer traits able to affect vectorial...

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Autores principales: Vitale, Matteo, Leo, Chiara, Courty, Thomas, Kranjc, Nace, Connolly, John B., Morselli, Giulia, Bamikole, Christopher, Haghighat-Khah, Roya Elaine, Bernardini, Federica, Fuchs, Silke
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081084/
https://www.ncbi.nlm.nih.gov/pubmed/35861105
http://dx.doi.org/10.1080/20477724.2022.2100192
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author Vitale, Matteo
Leo, Chiara
Courty, Thomas
Kranjc, Nace
Connolly, John B.
Morselli, Giulia
Bamikole, Christopher
Haghighat-Khah, Roya Elaine
Bernardini, Federica
Fuchs, Silke
author_facet Vitale, Matteo
Leo, Chiara
Courty, Thomas
Kranjc, Nace
Connolly, John B.
Morselli, Giulia
Bamikole, Christopher
Haghighat-Khah, Roya Elaine
Bernardini, Federica
Fuchs, Silke
author_sort Vitale, Matteo
collection PubMed
description The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in recent years sparked the development of transgenic technologies with the potential to be used as novel vector control tools. These technologies rely on genome editing that confer traits able to affect vectorial capacity. This can be achieved by either reducing the mosquito population or by making mosquitoes refractory to the parasite infection. For any genetically modified organism that is regarded for release, molecular characterization of the transgene and flanking sites are essential for their safety assessment and post-release monitoring. Despite great advancements, Whole-Genome Sequencing data are still subject to limitations due to the presence of repetitive and unannotated DNA sequences. Faced with this challenge, we describe a number of techniques that were used to identify the genomic location of a transgene in the male bias mosquito strain Ag(PMB)1 considered for potential field application. While the initial inverse PCR identified the most likely insertion site on Chromosome 3 R 36D, reassessment of the data showed a high repetitiveness in those sequences and multiple genomic locations as potential insertion sites of the transgene. Here we used a combination of DNA sequencing analysis and in-situ hybridization to clearly identify the integration of the transgene in a poorly annotated centromeric region of Chromosome 2 R 19D. This study emphasizes the need for accuracy in sequencing data for the genome of organisms of medical importance such as Anopheles mosquitoes and other tools available that can support genomic locations of transgenes.
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spelling pubmed-100810842023-04-08 Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes Vitale, Matteo Leo, Chiara Courty, Thomas Kranjc, Nace Connolly, John B. Morselli, Giulia Bamikole, Christopher Haghighat-Khah, Roya Elaine Bernardini, Federica Fuchs, Silke Pathog Glob Health Articles The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in recent years sparked the development of transgenic technologies with the potential to be used as novel vector control tools. These technologies rely on genome editing that confer traits able to affect vectorial capacity. This can be achieved by either reducing the mosquito population or by making mosquitoes refractory to the parasite infection. For any genetically modified organism that is regarded for release, molecular characterization of the transgene and flanking sites are essential for their safety assessment and post-release monitoring. Despite great advancements, Whole-Genome Sequencing data are still subject to limitations due to the presence of repetitive and unannotated DNA sequences. Faced with this challenge, we describe a number of techniques that were used to identify the genomic location of a transgene in the male bias mosquito strain Ag(PMB)1 considered for potential field application. While the initial inverse PCR identified the most likely insertion site on Chromosome 3 R 36D, reassessment of the data showed a high repetitiveness in those sequences and multiple genomic locations as potential insertion sites of the transgene. Here we used a combination of DNA sequencing analysis and in-situ hybridization to clearly identify the integration of the transgene in a poorly annotated centromeric region of Chromosome 2 R 19D. This study emphasizes the need for accuracy in sequencing data for the genome of organisms of medical importance such as Anopheles mosquitoes and other tools available that can support genomic locations of transgenes. Taylor & Francis 2022-07-21 /pmc/articles/PMC10081084/ /pubmed/35861105 http://dx.doi.org/10.1080/20477724.2022.2100192 Text en © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Articles
Vitale, Matteo
Leo, Chiara
Courty, Thomas
Kranjc, Nace
Connolly, John B.
Morselli, Giulia
Bamikole, Christopher
Haghighat-Khah, Roya Elaine
Bernardini, Federica
Fuchs, Silke
Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
title Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
title_full Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
title_fullStr Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
title_full_unstemmed Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
title_short Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
title_sort comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of anopheles mosquitoes
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081084/
https://www.ncbi.nlm.nih.gov/pubmed/35861105
http://dx.doi.org/10.1080/20477724.2022.2100192
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