Improving the Transposition Activity of Genie Plasmids

Abstract

The ability to stably integrate a gene of choice into mammalian genomes has applications in modern medical research and clinical therapies. Many studies involving the transfer of recombinant DNA, especially gene therapy research and transgenic animal production, require a vector that can quickly, efficiently, and safely direct the integration of genetic material into a genome in vivo. Transposon-mediated gene insertion is one of the most effective systems currently available for this purpose. In this study we designed and constructed pmGENIE-4, a novel vector for efficient mammalian transgenesis and gene therapy, based on the pmGENIE-3 vector created at the University of Hawaii Institute for Biogenesis Research. Unlike donor-helper vector systems that require co-transfection with a donor plasmid containing the transgene and a helper plasmid containing the transposase gene, pmGENIE-3 contains a single piggyBac transposase gene and transgene cassette on a single helper-independent plasmid. Another unique feature of pmGENIE-3 is that the transposase is self-inactivating after excision of the transgene from the plasmid. piggyBac excises the transgene cassette from the plasmid at terminal repeat elements (TREs), one of which is located in an intron of the piggyBac coding sequence. The transposase then inserts the fragment into TTAA sites in the host by the cut and paste mechanism typical of type II transposases. These self-inactivating constructs work well in transfecting cell lines and have proven competent for the production of transgenic mice. The piggyBac transposase is better suited for transgenesis than other transposases such as Sleeping Beauty, Tol2, and Mos1, due to its higher transpositional activity and larger cargo capacity. The pmGENIE-3 plasmid can also be converted into a BAC for large transgene insertions. To further improve upon the 2 transposition efficiency of the pmGENIE-3 vector, a new vector (pmGENIE-4) was constructed that carries two copies of the piggyBac gene. Two active transposases functioning simultaneously could potentially double the efficiency of the system, which is an important feature for applications such as transgenesis, where only a few copies of the plasmid may reach the nucleus. We successfully constructed the pmGENIE-4 plasmid and sequenced it to verify integrity. Quantitative real-time PCR (qRT-PCR) was performed and Western blot analysis will be performed to compare piggyBac mRNA and protein expression levels from pmGENIE-4 relative to pmGENIE-3. Finally, we will compare transpositional activities by colony count. If a higher transposition efficiency is achieved with the new plasmid, research in gene therapy and transgenesis will have a more effective tool at its disposal for the integration of recombinant DNA into host genomes.