Department of Developmental Biology, Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology, Göttingen Center of Molecular Biosciences, University of Göttingen, 37077 Göttingen, Germany; Göttingen Graduate Center for Neurosciences, Biophysics, and Molecular Biosciences, University of Göttingen, 37077 Goettingen, Germany; Molecular Cell Dynamics, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Department of Developmental Biology, Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology, Göttingen Center of Molecular Biosciences, University of Göttingen, 37077 Göttingen, Germany.
Insect Biochem Mol Biol. 2018 Jul;98:16-24. doi: 10.1016/j.ibmb.2018.04.001. Epub 2018 Apr 10.
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 (hyPBase) as well as in a mammalian codon-optimized (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 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.
即使在具有先进的靶向基因组编辑工具的时代，在转基因领域，DNA 转座酶的改进仍然有很高的需求：特别是在新兴的模式系统中，尚未创建评估的整合酶着陆位点，更重要的是在非模式生物中，如农业害虫和疾病载体，其中可靠的序列信息和基因组注释仍在等待中。事实上，随机插入诱变对于鉴定不受位置效应影响的新基因组位置至关重要，因此这些位置可以作为未来稳定的转基因整合位点。在这方面，已经设计了最广泛使用的 piggyBac 转座酶 (PBase) 的超活性版本。超活性版本 (hyPBase) 目前有原始昆虫密码子为基础的编码序列 (hyPBase) 以及哺乳动物密码子优化版本 (hyPBase)。与经典 PBase 相比，在类似的蛋白质水平下，这两种版本在哺乳动物体外和体内系统中表达时，转座的速度显著提高。在这里，我们证明了使用编码 hyPBase 的辅助质粒的用途 - 无论密码子使用情况如何 - 也显著提高了地中海实蝇 (Medfly) Ceratitis capitata、红面粉甲虫 Tribolium castaneum 和醋蝇 Drosophila melanogaster 的生殖系转化的成功率。因此，hyPBase 编码的辅助质粒非常适合生成不同昆虫目转基因品系。根据物种的不同，与 PBase 相比，我们实现了高达 15 倍的生殖系转化率，并生成了难以获得的影响适合度和生存力的转基因 T. castaneum 品系。此外，先前报道的由 hyPBase 编码的 hyPBase (iPB7) 引起的高不育率在我们的研究中无法得到证实。因此，我们认为 hyPBase 是一种有效的遗传工程工具，我们强烈推荐用于昆虫转基因。
