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Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas

BACKGROUND: The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this o...

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Autores principales: Cheng, Xi, Liu, Gai, Ke, Wenting, Zhao, Lijuan, Lv, Bo, Ma, Xiaocui, Xu, Nannan, Xia, Xiaoling, Deng, Xuan, Zheng, Chunlei, Huang, Kaiyao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430608/
https://www.ncbi.nlm.nih.gov/pubmed/28515773
http://dx.doi.org/10.1186/s13007-017-0183-5
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author Cheng, Xi
Liu, Gai
Ke, Wenting
Zhao, Lijuan
Lv, Bo
Ma, Xiaocui
Xu, Nannan
Xia, Xiaoling
Deng, Xuan
Zheng, Chunlei
Huang, Kaiyao
author_facet Cheng, Xi
Liu, Gai
Ke, Wenting
Zhao, Lijuan
Lv, Bo
Ma, Xiaocui
Xu, Nannan
Xia, Xiaoling
Deng, Xuan
Zheng, Chunlei
Huang, Kaiyao
author_sort Cheng, Xi
collection PubMed
description BACKGROUND: The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive. RESULTS: By selecting a suitable parental strain, keeping individual mutants using the agar plate, and designing an insertion cassette-specific primer for library screening, we successfully generated and maintained ~150,000 insertional mutants of Chlamydomonas, which was used for both reverse and forward genetics analysis. We obtained 26 individual mutants corresponding to 20 genes and identified 967 motility-defect mutants including 10 mutants with defective accumulation of intraflagellar transport complex at the basal body. We also obtained 929 mutants defective in oil droplet assembly after nitrogen deprivation. Furthermore, a new insertion cassette with splicing donor sequences at both ends was also constructed, which increased the efficiency of gene interruption. CONCLUSION: In summary, this library provides a multifunctional platform both for obtaining mutants of interested genes and for screening of mutants with specific phenotype. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-017-0183-5) contains supplementary material, which is available to authorized users.
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spelling pubmed-54306082017-05-17 Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas Cheng, Xi Liu, Gai Ke, Wenting Zhao, Lijuan Lv, Bo Ma, Xiaocui Xu, Nannan Xia, Xiaoling Deng, Xuan Zheng, Chunlei Huang, Kaiyao Plant Methods Methodology BACKGROUND: The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive. RESULTS: By selecting a suitable parental strain, keeping individual mutants using the agar plate, and designing an insertion cassette-specific primer for library screening, we successfully generated and maintained ~150,000 insertional mutants of Chlamydomonas, which was used for both reverse and forward genetics analysis. We obtained 26 individual mutants corresponding to 20 genes and identified 967 motility-defect mutants including 10 mutants with defective accumulation of intraflagellar transport complex at the basal body. We also obtained 929 mutants defective in oil droplet assembly after nitrogen deprivation. Furthermore, a new insertion cassette with splicing donor sequences at both ends was also constructed, which increased the efficiency of gene interruption. CONCLUSION: In summary, this library provides a multifunctional platform both for obtaining mutants of interested genes and for screening of mutants with specific phenotype. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-017-0183-5) contains supplementary material, which is available to authorized users. BioMed Central 2017-05-15 /pmc/articles/PMC5430608/ /pubmed/28515773 http://dx.doi.org/10.1186/s13007-017-0183-5 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology
Cheng, Xi
Liu, Gai
Ke, Wenting
Zhao, Lijuan
Lv, Bo
Ma, Xiaocui
Xu, Nannan
Xia, Xiaoling
Deng, Xuan
Zheng, Chunlei
Huang, Kaiyao
Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas
title Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas
title_full Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas
title_fullStr Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas
title_full_unstemmed Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas
title_short Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas
title_sort building a multipurpose insertional mutant library for forward and reverse genetics in chlamydomonas
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430608/
https://www.ncbi.nlm.nih.gov/pubmed/28515773
http://dx.doi.org/10.1186/s13007-017-0183-5
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