绵羊高效转基因通用型piggyBac转座子载体构建及功能验证

piggyBac (PB)是一种能在多种动物细胞中进行转座的DNA转座子,作为一种转基因工具已被广泛应用于各种哺乳动物转基因研究中。针对不同物种对PB转座子进行改造,是提升其通用性的必要手段。为构建基于绵羊细胞进行转基因操作的通用型PB转座子载体,本研究对PB转座酶(PBase)基因进行绵羊密码子偏好性优化并将其克隆到pBNW-TP1载体中,成功构建了PB转座子载体pBNW-TP2。将pBNW-TP2转染到绵羊成纤维细胞和乳腺上皮细胞中,利用G418筛选获取稳定转染细胞株;利用Tail-PCR检测稳定转染细胞株的PB转座位点,对细胞阳性克隆进行亚甲蓝染色;利用非配对t检验确认其转座效率。结果表明,pBNW-TP2成功介导了绵羊成纤维细胞和乳腺上皮细胞转基因阳性细胞株的生产;PB转座位点检测表明pBNW-TP2能特异性整合到绵羊基因组TTAA位点,其整合位点倾向于功能基因间;亚甲蓝染色统计分析结果提示pBNW-TP2介导的转基因效率显著提升。本研究成功构建了绵羊通用型PB转座子载体pBNW-TP2,并在绵羊体细胞中对其特性进行验证和分析,为PB转座子在绵羊体细胞中开展转基因相关研究提供了科学依据。

Exploration and characterization of a universal piggyBac transposon vector for efficient transgene studies in sheep

piggyBac (PB) is a DNA transposon that mediates transposition in various animal cells. As an efficient tool, PB transposons are applied to various transgenic research and optimized for different species, which is an essential means to enhance its versatility. To construct a universal PB transposase (PBase) vector for ovine transgene manipulation, the gene expression box of PBase with optimized bias for sheep codons was inserted into the pBNW-TP1 vector. The vector pBNW-TP2 with a single plasmid was successfully constructed. Then, pBNW-TP2 was transfected into ovine fibroblasts and mammary epithelial cells. The stable transfected cell lines were selected by G418 and the PB transposition sites were determined by Tail-PCR in the stable transfected cell lines. The transposition efficiency was verified by student’s t-tests for cell clones with methylene blue staining. The results showed that pBNW-TP2 successfully guided the production of transgenic ovine fibroblasts and mammary epithelial cell lines. The PB transposition test indicated that the pBNW-TP2 vector can be specifically integrated into the TTAA sites of the sheep genome. The statistical analysis of methylene blue staining results suggested that the transgenic efficiency mediated by the pBNW-TP2 vector increased significantly. In summary, we verified and analyzed characteristics of the universal PB transposon vector pBNW-TP2 for sheep in this study, which will provide a scientific basis for future transgenic research mediated by the PB transposon in ovine somatic cells.