Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 980-0845, Japan.
Fish Physiol Biochem. 2020 Jun;46(3):1121-1130. doi: 10.1007/s10695-020-00775-y. Epub 2020 Feb 23.
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.
我们用斑马鱼、牙鲆和水泡眼金鱼研究了电穿孔法将质粒递送至骨骼肌的效率，以及重组蛋白随后分泌到循环系统中的情况。电穿孔使 GFP 荧光的表达区域明显扩大。在斑马鱼中，引入的质粒保留在肌肉细胞中，并至少持续表达 GFP 180 天，表明质粒 DNA 在肌肉细胞中的寿命很长。荧光素酶和生长激素 (GH) 与荧光素酶的融合蛋白分别从用 CMV:secNluc 和 CMV:GH-Luc 质粒电穿孔的肌肉中分泌到血液中，表明肽类激素等重组蛋白可以通过质粒电穿孔递送至肌肉中的血液中。有趣的是，从用 CMV:secNluc 电穿孔的斑马鱼雌鱼产下的受精卵中检测到了荧光素酶活性，表明成熟的卵母细胞从肌肉中分泌的血液中摄取了重组蛋白。这里报道的质粒电穿孔系统也可能适用于将 Cas9 等重组蛋白递送至卵母细胞。由于在水泡眼金鱼的眼囊中检测到 GH-Luc 融合蛋白，这种鱼可能有助于生产重组蛋白。
