Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species
Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species
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DOI：
10.1016/j.ibmb.2018.04.001
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发表时间：
2018-07-01
影响因子：
3.8
通讯作者：
Wimmer, Ernst A.
中科院分区：
文献类型：
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作者：
Eckermann, Kolja N.;Ahmed, Hassan M. M.;Wimmer, Ernst A.
即使在先进的特定位点基因组编辑工具的时代，DNA转座酶的改进在转基因领域仍然有很高的需求：特别是在新兴的模式系统中，其中评估的整合酶着陆位点尚未创建，更重要的是在非模式生物中，如农业害虫和病媒，其中可靠的序列信息和基因组注释仍然悬而未决。事实上，随机插入突变对于确定不受位置效应影响的新的基因组位置是必不可少的，因此可以作为未来稳定的转基因整合位点。在这方面，已经设计出了最广泛使用的猪转座酶(Pbase)的超活性版本。超级活性版本(HyPBase)目前可用于原始的昆虫密码子编码序列((1)hyPBase)以及哺乳动物密码子优化的((M)hyPBase)版本。当在哺乳动物的体外和体内系统中表达时，这两种蛋白的转座率都比在类似蛋白水平上的经典PBase显著更高。在这里，我们证明了编码hyPBase的辅助质粒的使用-无论密码子的使用-也显著地增加了地中海果蝇(Medfly)Certis Capata、红粉甲虫Tribolium Castaneum和醋蝇DroSingila Blackogaster的种系转化成功率。因此，编码hyPbase的辅助子非常适合于产生不同昆虫目的转基因菌株。根据物种的不同，我们实现了比PBase高15倍的种系转化率，并产生了很难获得表达影响适应性和生存能力的结构的转基因卡氏锥虫菌株。此外，先前报道的由(1)hyPBase编码的hyPBase(IPB7)引起的高不孕率不能被我们的研究证实。因此，我们认为HyPBase是一种有效的基因工程工具，我们强烈推荐用于昆虫转基因。
Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence ((1)hyPBase) as well as in a mammalian codon-optimized ((m)hyPBase) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase- irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster. hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by (1)hyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis.