Plasmid delivery by electroporation into fish skeletal muscle for recombinant protein secretion and uptake by oocytes
Plasmid delivery by electroporation into fish skeletal muscle for recombinant protein secretion and uptake by oocytes
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DOI：
10.1007/s10695-020-00775-y
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发表时间：
2020-02-23
影响因子：
2.9
通讯作者：
Suzuki, Tohru
中科院分区：
文献类型：
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作者：
Iwaizumi, Masaki;Yokoi, Hayato;Suzuki, Tohru
我们使用斑马鱼、日本比目鱼和泡眼金鱼检查了电穿孔将质粒递送到骨骼肌中的效率，并检查了重组蛋白随后分泌到循环系统中的情况。电穿孔显着扩大了GFP荧光的表达区域。引入的质粒保留在斑马鱼的肌肉细胞中并持续表达 GFP 至少 180 天，表明质粒 DNA 在肌肉细胞中的寿命很长。荧光素酶以及生长激素（GH）和荧光素酶的融合物分别从用CMV:secNluc和CMV:GH-Luc质粒电穿孔的肌肉分泌到血液中，这表明重组蛋白如肽激素可以通过质粒电穿孔进入肌肉而供应到血液中。有趣的是，在用 CMV:secNluc 电穿孔的雌性斑马鱼产下的受精卵中检测到了荧光素酶活性，这表明成熟的卵母细胞掺入了来自肌肉分泌的血流的重组蛋白。这里报道的质粒电穿孔系统也可以用于将重组蛋白（例如 Cas9）递送到卵母细胞中。由于在泡眼金鱼的眼囊淋巴液中检测到了 GH-Luc 融合蛋白，因此这种鱼可能可用于生产重组蛋白。
We examined the efficiency of electroporation for the delivery of plasmid into the skeletal muscle and also examined the subsequent secretion of recombinant protein into the circulation system, using zebrafish, Japanese flounder, and bubble-eye goldfish. The expression area of GFP fluorescence was markedly expanded by electroporation. Introduced plasmid was retained in the muscle cells and continued to express GFP for at least 180 days in zebrafish, indicating the long lifespan of plasmid DNA in the muscle cell. Luciferase and a fusion of growth hormone (GH) and luciferase were secreted into the blood from muscle electroporated with CMV:secNluc and CMV:GH-Luc plasmids, respectively, indicating that recombinant proteins such as peptide hormones can be supplied to the blood by plasmid electroporation into muscle. Interestingly, luciferase activity was detected from fertilized eggs laid by zebrafish females that had been electroporated with CMV:secNluc, indicating that maturing oocytes incorporated recombinant protein from the blood stream that had been secreted from the muscle. The plasmid electroporation system reported here also may work for the delivery of recombinant proteins, such as Cas9, into the oocytes. Since the GH-Luc fusion protein was detected in the lymph of the eye-sac of bubble-eye goldfish, this fish may be useful for the production of recombinant protein.