The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.

The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.

将多基因构建体导入单细胞对于提高家畜的性能以及理解基本生物学过程非常重要。特别是，多基因构建体允许将与异种移植相关的多个基因工程化并整合到猪基因组中。PiggyBac（PB）转座子系统允许通过单次转染事件将多个基因稳定地整合到目标基因组中。然而，据我们所知，尚未有人尝试使用该系统将多个基因导入猪基因组。在本研究中，我们将7个转座子同时导入单个猪胚胎成纤维细胞（PEF）。用含有5种抗药蛋白基因和2种（红色和绿色）荧光蛋白基因的7个转座子以及一个PB转座酶表达载体pTrans转染PEF（实验组）。同时转染上述7个转座子（不含pTrans）作为对照组。在多种选择药物存在的情况下对这些转染细胞进行选择，结果实验组有几个克隆存活，而对照组没有。PCR分析表明，约90%（检测的13个中有12个）存活的克隆拥有所有导入的转座子。Splinkerette PCR表明转座子是通过PB的TTAA靶位点插入的。利用含有多基因构建体的PEF克隆进行体细胞核移植（SCNT），成功生产出表达红色和绿色荧光的克隆囊胚。这些结果表明这种PB介导的将多基因构建体同时转移到猪细胞基因组中的方法是可行的，这对于生产表达多个转基因的克隆转基因猪是有用的。