Highly efficient germ-line transmission of proviral insertions in zebrafish
Highly efficient germ-line transmission of proviral insertions in zebrafish
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DOI：
10.1073/pnas.93.15.7777
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发表时间：
1996-07-23
影响因子：
11.1
通讯作者：
Hopkins, N
中科院分区：
文献类型：
--
作者：
Gaiano, N;Allende, M;Hopkins, N
模式生物的一项重要技术是制造转基因动物的能力。在过去，斑马鱼转基因技术一直受到限制，相对较低的效率，转基因可以产生使用DNA显微注射或逆转录病毒感染。先前用逆转录病毒载体产生转基因斑马鱼的努力使用了假型病毒，其基因组基于莫洛尼鼠白血病病毒和水泡性口炎病毒的包膜蛋白。将这种病毒注射到囊胚期斑马鱼中，16%的注射胚胎将前病毒插入传递给后代，大多数创始人将单一插入传递给大约2%的后代。在努力提高这种转基因频率，我们已经产生了假型病毒股票的两个新的莫罗尼为基础的基因组。这些病毒储备液的滴度比以前使用的高两个数量级。这些病毒的注射导致转基因效率的显着增加;在三个不同的实验中，83%（110/133）的注射胚胎将前病毒插入传递给24%的后代。此外，用其中一种病毒制造的创始人通过其生殖系平均传播了11种不同的插入。这些结果代表了转基因斑马鱼的生产效率提高了50到100倍，使得现在一个实验室可以快速生产数万到数十万个转基因。因此，以前仅限于无脊椎动物的大规模插入诱变策略，现在可能在脊椎动物中。
An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximate to 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.