Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon
Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon
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DOI：
10.1016/j.ydbio.2003.07.013
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发表时间：
2003-11-15
影响因子：
2.7
通讯作者：
Ekker, SC
中科院分区：
文献类型：
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作者：
Davidson, AE;Balciunas, D;Ekker, SC
我们使用 Tc1/mariner 家族转座元件睡美人 (SB) 在斑马鱼 (Danio rerio) 中进行转基因和长期表达研究，斑马鱼是临床疾病、脊椎动物模式和细胞生物学应用的流行生物体。与标准的基于质粒注射的转基因方法相比，SB 转座酶将转基因和表达率提高了六倍（从 5% 提高到 31%），并将标记染色体总数增加了一倍多。对这些基因座的分子分析表明，这些元件精确整合到受体染色体中，并具有诊断转座的遗传足迹。转座酶介导的整合体的 GFP 表达在第八代中是孟德尔式的。蓝移 GFP 变体 (BFP) 和红色荧光蛋白 (DsRed) 也是有用的转基因标记，表明多个报告基因可在斑马鱼中与 SB 一起使用。我们证明 SB 适合使用缩写的 γ-晶状体蛋白 GFP 盒进行组织特异性转基因应用。最后，我们描述了一种用于斑马鱼染色体工程和分子遗传学实验的通用转座子载体。总之，这些数据表明 SB 是斑马鱼转基因和表达的有效工具，并且转座子将可用于细胞生物学应用中的基因表达以及发育过程中基因发现的插入诱变剂。 (C) 2003 Elsevier Inc. 保留所有权利。
We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development. (C) 2003 Elsevier Inc. All rights reserved.