转基因沉默通过使用病毒载体或转座子的基因工程在动物模型生产中提供了重大挑战。选择合适的策略,取决于物种对于避免转基因沉默至关重要,需要长期观察体内基因表达。该研究使用PiggyBac转座子来创建GFP大鼠模型以解决大鼠中的转基因沉默。令人惊讶的是,使用CAG启动子时发生转基因沉默,与传统理解相反,而Ef1α启动子阻止沉默。GFP表达在五代以上保持稳定,证实Ef1α启动子对大鼠长期蛋白表达的功效。此外,GFP表达在由GFP大鼠产生的各种细胞来源中始终维持在细胞水平。从而验证GFP大鼠的体外GFP表达。全基因组测序在Akap1外显子1和2之间确定了一个稳定的整合位点,减轻了序列无关机制介导的转基因沉默。本研究建立了一种使用PiggyBac转座子产生转基因大鼠模型的有效方法。我们的GFP大鼠代表了第一个在五代中表现出外源基因延长表达的模型,对基因工程大鼠模型的未来研究具有重要意义。
Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.