Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System
نویسندگان
چکیده
Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.
منابع مشابه
High-level transgene expression mediated by the piggyBac transposon enhances transgenic therapeutic effects in cervical cancer xenografts.
The piggyBac (PB) transposon is a recently identified, active and flexible transgene vector, combining the advantages of non-viral gene delivery with genomic integration and persistent transgene expression. In this study, we utilized the PB transposon to carry the herpes simplex thymidine kinase (HSV-tk) and red fluorescent protein (mRFP1) reporter genes into the HeLa cervical cancer cell line ...
متن کاملHyperactive piggyBac gene transfer in human cells and in vivo.
We characterized a recently developed hyperactive piggyBac (pB) transposase enzyme [containing seven mutations (7pB)] for gene transfer in human cells in vitro and to somatic cells in mice in vivo. Despite a protein level expression similar to that of native pB, 7pB significantly increased the gene transfer efficiency of a neomycin resistance cassette transposon in both HEK293 and HeLa cultured...
متن کاملDelivery of Full-Length Factor VIII Using a piggyBac Transposon Vector to Correct a Mouse Model of Hemophilia A
Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on pi...
متن کاملMultiplexed transposon-mediated stable gene transfer in human cells.
Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonvir...
متن کامل