{"title":"具有晶状体特异性 Cre 表达的转基因斑马鱼品系的产生和特征。","authors":"Xuyan Peng, Xiaolin Jia, Guohui Shang, Mengjiao Xue, Mingjun Jiang, Dandan Chen, Fengyan Zhang, Yanzhong Hu","doi":"","DOIUrl":null,"url":null,"abstract":"<p><strong>Purpose: </strong>Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens.</p><p><strong>Methods: </strong>The pTol2 <i>cryaa</i>:Cre-polyA-<i>cryaa</i>:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish.</p><p><strong>Results: </strong>In this study, we generated a transgenic zebrafish line, zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific <i>cryaa</i> promoter. zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) embryos were injected with the <i>loxP</i>-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses.</p><p><strong>Conclusions: </strong>We established a zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.</p>","PeriodicalId":18866,"journal":{"name":"Molecular Vision","volume":"30 ","pages":"123-136"},"PeriodicalIF":1.8000,"publicationDate":"2024-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11006009/pdf/","citationCount":"0","resultStr":"{\"title\":\"The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression.\",\"authors\":\"Xuyan Peng, Xiaolin Jia, Guohui Shang, Mengjiao Xue, Mingjun Jiang, Dandan Chen, Fengyan Zhang, Yanzhong Hu\",\"doi\":\"\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Purpose: </strong>Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens.</p><p><strong>Methods: </strong>The pTol2 <i>cryaa</i>:Cre-polyA-<i>cryaa</i>:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish.</p><p><strong>Results: </strong>In this study, we generated a transgenic zebrafish line, zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific <i>cryaa</i> promoter. zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) embryos were injected with the <i>loxP</i>-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses.</p><p><strong>Conclusions: </strong>We established a zTg(<i>cryaa</i>:Cre-<i>cryaa</i>:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.</p>\",\"PeriodicalId\":18866,\"journal\":{\"name\":\"Molecular Vision\",\"volume\":\"30 \",\"pages\":\"123-136\"},\"PeriodicalIF\":1.8000,\"publicationDate\":\"2024-03-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11006009/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Vision\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Vision","FirstCategoryId":"3","ListUrlMain":"","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression.
Purpose: Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens.
Methods: The pTol2 cryaa:Cre-polyA-cryaa:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish.
Results: In this study, we generated a transgenic zebrafish line, zTg(cryaa:Cre-cryaa:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific cryaa promoter. zTg(cryaa:Cre-cryaa:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(cryaa:Cre-cryaa:EGFP) embryos were injected with the loxP-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(cryaa:Cre-cryaa:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses.
Conclusions: We established a zTg(cryaa:Cre-cryaa:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.
期刊介绍：
Molecular Vision is a peer-reviewed journal dedicated to the dissemination of research results in molecular biology, cell biology, and the genetics of the visual system (ocular and cortical).
Molecular Vision publishes articles presenting original research that has not previously been published and comprehensive articles reviewing the current status of a particular field or topic. Submissions to Molecular Vision are subjected to rigorous peer review. Molecular Vision does NOT publish preprints.
For authors, Molecular Vision provides a rapid means of communicating important results. Access to Molecular Vision is free and unrestricted, allowing the widest possible audience for your article. Digital publishing allows you to use color images freely (and without fees). Additionally, you may publish animations, sounds, or other supplementary information that clarifies or supports your article. Each of the authors of an article may also list an electronic mail address (which will be updated upon request) to give interested readers easy access to authors.
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