Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct
Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct
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DOI：
10.1038/nprot.2006.165
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Wittbrodt, Joachim
中科院分区：
文献类型：
--
作者：
Rembold, Martina;Lahiri, Kajori;Wittbrodt, Joachim
小型鱼类是通过转基因研究基因表达和功能的流行实验室模型。然而，如果转基因不容易通过目视检查检测到，则必须注射大量胚胎，进行饲养和筛选以识别阳性创始鱼。在这里，我们描述了一种策略，以有效地产生和预选转基因系携带任何感兴趣的转基因。共注射可选择的报告构建体（例如，GFP）与使用I-SceI大范围核酸酶方法在单独质粒上的目的转基因一起，导致两种质粒的共分布。因此，F0代内GFP表达的质量反映了注射的质量，并允许有效和可靠地选择对第二目的转基因也呈阳性的创始鱼。根据我们的经验，大部分（高达50%）GFP阳性鱼也将是第二转基因的转基因鱼，从而提供了一种快速（3-4个月内）和有效的方法来建立青鳉和斑马鱼中任何感兴趣基因的转基因品系。
Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish.