Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange
Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange
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DOI：
10.1089/clo.2008.0024
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发表时间：
2008-09-01
期刊：
影响因子：
--
通讯作者：
Nagashima, Hiroshi
中科院分区：
文献类型：
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作者：
Matsunari, Hitomi;Onodera, Masafumi;Nagashima, Hiroshi
带有荧光蛋白等细胞标记的基因工程猪在包括跟踪移植细胞或组织在内的研究中非常有用。在这项研究中，我们生产了转基因克隆猪携带的基因，新开发的红色荧光蛋白，人源化Kusabira橙（huKO），这是克隆的珊瑚石真菌concinna。用逆转录病毒载体D Delta Nsap转染huKO cDNA的胎儿成纤维细胞重构核移植胚胎，体外发育率为28.0%（37/132）。几乎所有（94.6%，35/37）的来自转导细胞的克隆胚泡表现出明确的huKO基因表达。共有429个核移植胚胎被转移到4个受体，所有受体都怀孕了，总共生下了18个转基因克隆后代。所有猪在分析的所有23个器官和组织中高度表达huKO荧光，包括脑、眼、肠和生殖器官、骨骼肌、骨、皮肤和蹄。此外，这种表达还通过各种组织如胰岛、肾小体、神经元和神经胶质细胞、视网膜、软骨细胞和造血细胞的组织学分析来证实。这些数据表明，表现出系统性红色荧光表达的转基因克隆猪可以通过用huKO基因逆转录病毒转导的体细胞的核转移有效地产生。
Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector D Delta Nsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.