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Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs
Qiu, Chao1,2; Cheng, Bin3; Zhang, Yunsheng1; Huang, Rong1; Liao, Lanjie1; Li, Yongming1; Luo, Daji3; Hu, Wei1; Wang, Yaping1,2
2014-12-01
Source PublicationMARINE BIOTECHNOLOGY
ISSN1436-2228
Volume16Issue:6Pages:674-683
AbstractTranscription activator-like effector nucleases (TALENs) are used for gene knockout and genome-editing studies in zebrafish, and these techniques have the potential to be applied to other fish species. Here, we show that TALENs can directly knock out a green fluorescent protein (GFP) transgene in medaka by affecting translation and synthesis of the GFP. We constructed a transgenic plasmid (pGFP-RFP) carrying the GFP and red fluorescent protein (RFP) genes, and used a modified TALEN method to assemble a pair of TALENs for the core chromophore Y66 region of GFP. Embryo toxicity of TALEN messenger RNA (mRNA) was far lower than the linearized plasmid; meanwhile, 76.3 % embryos, green fluorescence of embryos decreased significantly after co-injection of TALEN mRNA and the linearized plasmid, but red fluorescence showed no significant change. Real-time quantitative polymerase chain reaction and sequencing results showed that nearly 100 % mutated GFP position was disrupted at the Y66 region of GFP in the coinjected medaka embryos, caused by TALENs. This led to random insertion-deletion of nucleotides, which affected the translation of GFP and disrupted GFP synthesis. This provides new experimental evidence for designing TALEN sites in genes for which only key functional domains are known. Our results show that a modified TALEN method can efficiently and specifically mediate a transgene knockout in medaka. This report may promote the application of TALENs in gene-editing studies of fish species other than zebrafish.
SubtypeArticle
KeywordMedaka Talen Green Fluorescent Protein Gene
DOI10.1007/s10126-014-9584-x
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
Indexed BySCI
Language英语
WOS Research AreaBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
WOS SubjectBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
WOS IDWOS:000344543800005
WOS KeywordZINC-FINGER NUCLEASES ; EFFECTOR NUCLEASES ; ORYZIAS-LATIPES ; ZEBRAFISH ; DISRUPTION ; EMBRYOS ; FISH ; DOMAIN ; CELLS ; DMY
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Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/20326
Collection鱼类生物学及渔业生物技术研究中心_期刊论文
Affiliation1.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100039, Peoples R China
3.Wuhan Univ, Sch Basic Med Sci, Dept Genet, Wuhan 430071, Hubei, Peoples R China
Recommended Citation
GB/T 7714
Qiu, Chao,Cheng, Bin,Zhang, Yunsheng,et al. Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs[J]. MARINE BIOTECHNOLOGY,2014,16(6):674-683.
APA Qiu, Chao.,Cheng, Bin.,Zhang, Yunsheng.,Huang, Rong.,Liao, Lanjie.,...&Wang, Yaping.(2014).Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs.MARINE BIOTECHNOLOGY,16(6),674-683.
MLA Qiu, Chao,et al."Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs".MARINE BIOTECHNOLOGY 16.6(2014):674-683.
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