Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
Biochimie. 2011 Oct;93(10):1858-64. doi: 10.1016/j.biochi.2011.07.006. Epub 2011 Jul 18.
As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.
由于斑马鱼是遗传学、发育生物学和毒理学中常用的鱼类模式生物,因此开发一种高效的斑马鱼转基因技术对于各种生物学实验都非常重要。在这里,我们证明了玉米转座子系统 Ac/Ds 极大地提高了显微注射 DNA 的转基因效率。利用 Ac/Ds 系统,我们成功地建立了两个具有生殖系传递的稳定的转基因斑马鱼系,Tg(hsp70:gfp)和 Tg(cyp1a1:gfp),其生殖系传递效率分别为 83.3%(10/12)和 100.0%(4/4)。来自不同转基因系的 F1 代中,转基因后代的比例在 3.1%至 100.0%之间。有趣的是,我们从转基因系中发现了多个插入,并通过克隆插入位点证实了 Ac 转座酶介导的转座。此外,我们还证明了这两个 GFP 转基因斑马鱼系都能诱导 GFP 表达。在 Tg(hsp70:gfp)中,GFP 基因受热休克诱导的斑马鱼 hsp70 启动子控制,热休克后 GFP 广泛表达。在 Tg(cyp1a1:gfp)中,GFP 基因由可被 2,3,7,8-四氯二苯并二恶英(TCDD)等多种外源化学物质激活的斑马鱼 cyp1a1 启动子驱动;事实上,TCDD 能诱导 GFP 在肝脏、肠道和肾脏中表达。我们的数据表明,在玉米 Ac/Ds 转座子系统的辅助下,实现了高效的转基因。