可同时表达4种荧光蛋白克隆猪问世
记者在中科院广州生物医药与健康研究院获悉，该院近期成功培育出一种特殊的转基因克隆猪，这种克隆猪在特定波长的激发光下可分别发出红、黄、绿、青4种荧光，这是国际上首次获得能够同时表达4种荧光蛋白的转基因克隆猪。
据研究人员介绍，该项成果的运用，将极大地促进含多种优良性状的转基因克隆猪的研究与产业化，为今后加速我国对猪的遗传性状进行转基因改良，提高其生产性能，制作用于人类疾病的动物模型以及作为人类器官移植供体的转基因猪研究奠定基础。
该研究是广州生物医药与健康研究院研究员赖良学团队，与南方医科大学教授顾为望及华南农业大学教授吴珍芳合作完成的。研究组通过电穿孔的方法分别将红色、黄色荧光蛋白、绿色以及青色荧光蛋白的4种基因同时转入长白猪胎儿成纤维细胞，然后进行体细胞克隆，将克隆胚胎移入母猪体内，最终获得11头转基因克隆猪。
目前，转基因克隆猪已健康成长一年多，并开始繁殖下一代。该研究成果于日前发表于国际著名期刊《公共科学图书馆—综合》（PLoS ONE）上。
体细胞克隆技术是目前生产转基因大动物最有效的途径，但受体细胞增殖能力有限的影响，此前的研究中，每一轮克隆只能转入一个外源基因。赖良学团队经过潜心研究，利用2A短肽序列巧妙地将不同的荧光蛋白基因连在同一个载体上，只通过一轮动物克隆即成功获得了含多个外源转基因的克隆猪，不仅克服了体细胞多基因转移的技术障碍，而且实现了多基因在高水平上的协同表达。
猪是人类组织、细胞和器官异种移植最有前途的供体，克隆猪表达的荧光蛋白为追踪猪组织、细胞和器官在受者体内存活和机能状况提供了重要的标识，表达4种荧光蛋白的克隆猪将成为异种移植研发的理想动物模型。（生物谷Bioon.com）
PLoS ONE DOI:10.1371/journal.pone.0019986.g001
Use of the 2A Peptide for Generation of Multi-Transgenic Pigs through a Single Round of Nuclear Transfer
Wei Deng, Dongshan Yang, Bentian Zhao, Zhen Ouyang, Jun Song, Nana Fan, Zhaoming Liu, Yu Zhao, Qinghong Wu, Bayaer Nashun, Jiangjing Tang, Zhenfang Wu, Weiwang Gu, Liangxue Lai
Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.
