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Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells
A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technolog...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582328/ https://www.ncbi.nlm.nih.gov/pubmed/31062017 http://dx.doi.org/10.1093/nar/gkz343 |
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author | Asoshina, Manami Myo, Genki Tada, Natsuko Tajino, Koji Shimizu, Noriaki |
author_facet | Asoshina, Manami Myo, Genki Tada, Natsuko Tajino, Koji Shimizu, Noriaki |
author_sort | Asoshina, Manami |
collection | PubMed |
description | A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage–fusion–bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology. |
format | Online Article Text |
id | pubmed-6582328 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-65823282019-06-21 Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells Asoshina, Manami Myo, Genki Tada, Natsuko Tajino, Koji Shimizu, Noriaki Nucleic Acids Res Synthetic Biology and Bioengineering A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage–fusion–bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology. Oxford University Press 2019-06-20 2019-05-07 /pmc/articles/PMC6582328/ /pubmed/31062017 http://dx.doi.org/10.1093/nar/gkz343 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://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/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Synthetic Biology and Bioengineering Asoshina, Manami Myo, Genki Tada, Natsuko Tajino, Koji Shimizu, Noriaki Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells |
title | Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells |
title_full | Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells |
title_fullStr | Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells |
title_full_unstemmed | Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells |
title_short | Targeted amplification of a sequence of interest in artificial chromosome in mammalian cells |
title_sort | targeted amplification of a sequence of interest in artificial chromosome in mammalian cells |
topic | Synthetic Biology and Bioengineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582328/ https://www.ncbi.nlm.nih.gov/pubmed/31062017 http://dx.doi.org/10.1093/nar/gkz343 |
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