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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...

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Autores principales: Asoshina, Manami, Myo, Genki, Tada, Natsuko, Tajino, Koji, Shimizu, Noriaki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
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.
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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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