RESUMO
Efficient transgene delivery is critical for genetic manipulation and therapeutic intervention of target cells. Two well-characterized integrative systems have been described that rely on viral and nonviral vectors. However, use of viral vectors for gene therapy has been associated with several safety concerns. Here, we report a virus-free method for stable transgenesis based on the reaction of retroviral integrase. We constructed a gateway cloning compatible vector containing two truncated long terminal repeat (LTR) sequences (dLTR) that flank the transgene cassette. Notably, 5'-ACTG-3' and blunt-end restriction cutting sites were also embedded at the end of dLTR to be recognized by HIV-1 integrase. When performing coinjection of transgene cassette and integrase mRNA into zebrafish embryos at one cell stage, there were 50% to 55% of injected embryos expressing a marker gene in a desired pattern. When applying our method in mammalian cells, there were 42% of cultured human epithelial cell lines showing stable integration. These results demonstrated that our method can successfully insert an exogenous gene into the host genome with highly efficient integration. Importantly, this system operates without most of the viral components while retaining effective stable transgenesis. We anticipate this method will provide a convenient, safe, and highly efficient way for applications in transgenesis and gene therapy.
