Institute of Biotechnology and Department of Fundamental Microbiology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Lausanne, Switzerland.
UMR INRAE 0085, CNRS 7247, Physiologie de la Reproduction et des Comportements, Centre INRAE Val de Loire, 37380 Nouzilly, France.
J Biotechnol. 2021 Nov 20;341:103-112. doi: 10.1016/j.jbiotec.2021.09.011. Epub 2021 Sep 21.
Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.
整合非病毒载体，如经过工程改造以介导靶向基因转移到基因组中的安全港位点的转座子，可能是一种有前途的方法，可用于高效和安全地生产治疗性蛋白质或进行基因治疗。在这方面，我们设计并评估了两种靶向核核糖体 DNA（rDNA）基因座的策略。一种方法依赖于使用核仁定位信号（NoLS）使转座酶和转座子与转录活跃的 rDNA 拷贝共定位。另一种方法包括使用 NoLS-FokI-dCas9 内切酶靶向 rDNA 基因座中的 18S 编码区，以进行靶向转基因敲入。我们表明，使用 piggyBac 转座子系统可以以高频率将整合到中国仓鼠卵巢（CHO）细胞的 rDNA 中，这表明 rDNA 非常容易进行转座。一致地，当 piggyBac 转座子 DNA 和转座酶都靶向核仁时，最常获得 rDNA 靶向转座事件，产生稳定且均匀表达转基因的细胞。因此，该方法为使用 CHO 细胞提高靶向转基因传递和蛋白表达提供了一种替代策略。
