Institute for Biogenesis Research, Department of Anatomy, Biochemistry, and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
Nucleic Acids Res. 2012 Aug;40(14):6978-91. doi: 10.1093/nar/gks309. Epub 2012 Apr 9.
Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.
整合载体，如病毒和转座子，将转基因随机插入，可能会破坏或调节基因。为了使这些技术具有治疗价值，需要一种控制插入精确位置的方法。piggyBac(PB)转座酶是一种有效的基因转移载体，在多种细胞类型中均具有活性，并已被证明可进行修饰。在这里，我们设计并验证了融合到 Gal4 DNA 结合域的嵌合 PB 蛋白，其能够将转基因靶向预定的位置。在人细胞中，带有上游激活序列(UAS)Gal4 识别位点的受体质粒被嵌合 Gal4-PB 转座酶优先靶向。为了分析这些 PB 融合蛋白靶向染色体位置的能力，使用 Sleeping Beauty 转座子将 UAS 位点随机整合到基因组中。N-和 C-末端 Gal4-PB 融合蛋白但不是天然 PB 均能够靶向这些引入的位点附近的转座。全基因组整合分析表明，我们的融合构建体能够使 24%的整合偏向于内源性 Gal4 识别序列附近。这项工作为增强 PB 系统在基因工程和基因治疗等应用中的特性提供了一种强大的方法。
