Gaiano N, Allende M, Amsterdam A, Kawakami K, Hopkins N
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7777-82. doi: 10.1073/pnas.93.15.7777.
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 approximately 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.
在模式生物中，一项重要技术是制造转基因动物的能力。过去，斑马鱼中的转基因技术受到限制，因为使用DNA显微注射或逆转录病毒感染来产生转基因的效率相对较低。以前使用逆转录病毒载体生成转基因斑马鱼的努力中，使用了一种假型病毒，其基因组基于莫洛尼鼠白血病病毒，包膜蛋白来自水疱性口炎病毒。这种病毒被注射到囊胚期的斑马鱼中，16%的注射胚胎将前病毒插入传递给了它们的后代，大多数奠基者将单个插入传递给了大约2%的后代。为了提高这种转基因频率，我们构建了两种基于莫洛尼病毒的新基因组的假型病毒库。这些病毒库的滴度比以前使用的高出两个数量级。注射这些病毒导致转基因效率大幅提高；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了24%的后代。此外，用其中一种病毒产生的奠基者通过其生殖系平均传递了11种不同的插入。这些结果表明，生成转基因斑马鱼的效率提高了50到100倍，使得单个实验室现在能够快速生成数万到数十万的转基因成为可能。因此，以前仅限于无脊椎动物的大规模插入诱变策略现在在脊椎动物中也可能实现。
