Commonly used viral vectors have been shown to preferentially insert their cargo near transcriptional start sites ( 1–3 ) and there has been increasing concern for the implications of insertional mutagenesis ( 4–6 ). For these techniques to be of value in the clinical setting it is imperative that insertions occur at known safe loci in order to avoid deregulation of the cell due to deleterious integrations and to control expression of transgenes. The ability of integrating vectors to permanently introduce foreign genes into chromosomes has resulted in major advances in the fields of genetic engineering, functional genomics and gene therapy. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences.
Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. 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. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4–PB transposase in human cells. 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. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes.