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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
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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. |
format | Online Article Text |
id | pubmed-5430608 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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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