Plasmid delivery by electroporation into fish skeletal muscle for recombinant protein secretion and uptake by oocytes


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.

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  1. Babaei F, Ramalingam R, Tavendale A, Liang Y, Yan LS, Ajuh P et al (2013) Novel blood collection method allows plasma proteome analysis from single zebrafish. J Proteome Res 12(4):1580–1590.

    CAS  Article  PubMed  Google Scholar 

  2. Callahan SJ, Tepan S, Zhang YM, Lindsay H, Burger A, Campbell NR et al (2018) Cancer modeling by transgene electroporation in adult zebrafish (TEAZ). Dis Model Mech 11(9).

  3. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Lee LYY, Izzard L, Hurt AC (2018) A review of DNA vaccines against influenza. Front Immunol 9:1568.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Li A, Sadasivam M, Ding JL (2003) Receptor-ligand interaction between vitellogenin receptor (VtgR) and vitellogenin (Vtg), implications on low density lipoprotein receptor and apolipoprotein B/E. The first three ligand-binding repeats of VtgR interact with the amino-terminal region of Vtg. J Biol Chem 278(5):2799–2806.

    CAS  Article  PubMed  Google Scholar 

  6. Lv W, Jiang P, Wang W, Wang X, Wang K, Chang L et al (2018) Electrotransfer of single-chain LH gene into skeletal muscle induces early ovarian development of orange-spotted grouper (Epinephelus coioides). Gen Comp Endocrinol 259:12–19.

    CAS  Article  PubMed  Google Scholar 

  7. Matsuda Y, Ito Y, Hashimoto H, Yokoi H, Suzuki T (2011) Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence. Reprod Biol Endocrinol 9:45.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Myllymaki H, Niskanen M, Oksanen K, Ramet M (2018) Immunization of adult zebrafish for the preclinical screening of DNA-based vaccines. J Vis Exp 140.

  9. Nakamura H, Katahira T, Sato T, Watanabe Y, Funahashi J (2004) Gain- and loss-of-function in chick embryos by electroporation. Mech Dev 121(9):1137–1143.

    CAS  Article  PubMed  Google Scholar 

  10. Rambabu KM, Rao SH, Rao NM (2005) Efficient expression of transgenes in adult zebrafish by electroporation. BMC Biotechnol 5:29.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Sawatari E, Hashimoto H, Matsumura T, Iwata Y, Yamamoto N, Yokoyama Y, Wakamatsu Y (2009) Cell growth-promoting activity of fluid from eye sacs of the bubble-eye goldfish (Carassius auratus). Zool Sci 26(4):254–258.

    Article  PubMed  Google Scholar 

  12. Sokolowska E, Blachnio-Zabielska AU (2019) A critical review of electroporation as a plasmid delivery system in mouse skeletal muscle. Int J Mol Sci 20(11).

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This work was supported by grants from the Japan Ministry of Education, Culture, Sports, Science, and Technology (grant numbers 18J20434 to MI; 19K22323 and 17H03867 to TS; and 18K05813 and 15KK0272 to HY) and the Program for Creation of Interdisciplinary Research from the Frontier Research Institute for Interdisciplinary Sciences, Tohoku University (to HY). Iwaizumi is a Research Fellow of Japan Society for the Promotion of Science.

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Correspondence to Tohru Suzuki.

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Iwaizumi, M., Yokoi, H. & Suzuki, T. Plasmid delivery by electroporation into fish skeletal muscle for recombinant protein secretion and uptake by oocytes. Fish Physiol Biochem 46, 1121–1130 (2020).

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  • Luciferase
  • GFP
  • Ovary
  • Zebrafish
  • Flounder
  • Goldfish