A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish
Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period...
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2021-08-01
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Online Access: | https://www.mdpi.com/2079-7737/10/8/777 |
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doaj-fecd519965004c7da55d9f2bac77298d |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Chong Zhang Jiaguang Li Imran Tarique Yizhuang Zhang Tong Lu Jiasheng Wang Aijun Chen Fenfen Wen Zhuoyu Zhang Yanjun Zhang Ming Shao |
spellingShingle |
Chong Zhang Jiaguang Li Imran Tarique Yizhuang Zhang Tong Lu Jiasheng Wang Aijun Chen Fenfen Wen Zhuoyu Zhang Yanjun Zhang Ming Shao A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish Biology maternal factors oocyte CRISPR/Cas9 double mutant early development zebrafish |
author_facet |
Chong Zhang Jiaguang Li Imran Tarique Yizhuang Zhang Tong Lu Jiasheng Wang Aijun Chen Fenfen Wen Zhuoyu Zhang Yanjun Zhang Ming Shao |
author_sort |
Chong Zhang |
title |
A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish |
title_short |
A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish |
title_full |
A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish |
title_fullStr |
A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish |
title_full_unstemmed |
A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish |
title_sort |
time-saving strategy to generate double maternal mutants by an oocyte-specific conditional knockout system in zebrafish |
publisher |
MDPI AG |
series |
Biology |
issn |
2079-7737 |
publishDate |
2021-08-01 |
description |
Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(<i>zpc</i>:<i>zcas9</i>) with oocyte-specific <i>cas9</i> expression, we have successfully obtained maternal or maternal–zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target <i>dvl2</i> and <i>dvl3a</i> simultaneously and introduced this construct to the genome of Tg(<i>zpc</i>:<i>zcas9</i>) by meganuclease I-<i>Sce</i> I. As expected, we confirmed the existence of M<i>dvl2</i>;M<i>dvl3a</i> embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZ<i>dvl2</i>;MZ<i>dvl3a</i> embryos were also obtained by crossing the mutant carrying mosaic F0 female with <i>dvl2</i><sup>+/−</sup>;<i>dvl3a</i><sup>−/−</sup> male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs. |
topic |
maternal factors oocyte CRISPR/Cas9 double mutant early development zebrafish |
url |
https://www.mdpi.com/2079-7737/10/8/777 |
work_keys_str_mv |
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doaj-fecd519965004c7da55d9f2bac77298d2021-08-26T13:32:17ZengMDPI AGBiology2079-77372021-08-011077777710.3390/biology10080777A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in ZebrafishChong Zhang0Jiaguang Li1Imran Tarique2Yizhuang Zhang3Tong Lu4Jiasheng Wang5Aijun Chen6Fenfen Wen7Zhuoyu Zhang8Yanjun Zhang9Ming Shao10Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaTaishan College, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaTaishan College, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaShandong Provincial Key Laboratory of Animal Cell and Developmental Biology and Key Laboratory for Experimental Teratology of the Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, ChinaMaternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(<i>zpc</i>:<i>zcas9</i>) with oocyte-specific <i>cas9</i> expression, we have successfully obtained maternal or maternal–zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target <i>dvl2</i> and <i>dvl3a</i> simultaneously and introduced this construct to the genome of Tg(<i>zpc</i>:<i>zcas9</i>) by meganuclease I-<i>Sce</i> I. As expected, we confirmed the existence of M<i>dvl2</i>;M<i>dvl3a</i> embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZ<i>dvl2</i>;MZ<i>dvl3a</i> embryos were also obtained by crossing the mutant carrying mosaic F0 female with <i>dvl2</i><sup>+/−</sup>;<i>dvl3a</i><sup>−/−</sup> male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs.https://www.mdpi.com/2079-7737/10/8/777maternal factorsoocyteCRISPR/Cas9double mutantearly developmentzebrafish |