Bire Solenne, Ley Déborah, Casteret Sophie, Mermod Nicolas, Bigot Yves, Rouleux-Bonnin Florence
GICC, UMR CNRS 7292, Université François Rabelais, Tours, France ; Institute of Biotechnology, University of Lausanne, and Center for Biotechnology UNIL-EPFL, Lausanne, Switzerland ; PRC, UMR INRA-CNRS 7247, Centre INRA Val de Loire, Nouzilly, France.
PLoS One. 2013 Dec 3;8(12):e82559. doi: 10.1371/journal.pone.0082559. eCollection 2013.
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.
将转基因稳定整合并表达于细胞基因组中，对于基于基因的治疗和生物生产目的而言，仍然是重大挑战。虽然转座子载体可介导高效的转基因整合，但其表达可能会受到表观遗传沉默的限制，并且持续的转座酶表达可能会介导多个转座循环。在此，我们评估了与基因绝缘转座子相结合的猪尾巴（PiggyBac）转座酶信使核糖核酸（mRNA）的递送，以将转基因与邻近调控元件分离并稳定表达。我们比较了信使核糖核酸和DNA载体中猪尾巴转座酶的表达情况，涉及表达水平、转座效率、转基因表达和基因毒性效应，以便校准并确保基于转座的递送系统。信使核糖核酸将转座酶的持续存在时间缩短至一个狭窄窗口，从而减少了诸如多余基因组DNA切割等副作用。发现CTF/NF1和D4Z4绝缘子均能从少数转座事件中介导更高效的表达。我们得出结论，使用工程化的猪尾巴转座酶信使核糖核酸和绝缘转座子为提高整合过程质量和维持转座子载体表达提供了有前景的方法。
