Journal of Zhejiang University-SCIENCE B

, Volume 20, Issue 12, pp 1021–1026 | Cite as

Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via l-arginine deprivation in HEK-293 cells

  • Zi-fei Yin
  • Ya-ni Zhang
  • Shu-fang Liang
  • Sha-sha Zhao
  • Juan Du
  • Bin-bin ChengEmail author

支原体污染通过耗竭l-精氨酸降低HEK-293 细 胞中质粒DNA 转染效率


目 的

明确支原体污染对HEK-293 细胞质粒DNA 转染 效率的影响, 并从支原体对细胞精氨酸代谢的角 度探究其机制。


支原体是细胞培养中的常见污染源。HEK-293 是 目前常用的生产蛋白、包装病毒的常用细胞系。 然而, 目前支原体污染对于质粒DNA 转染效率 的影响未见报道。本研究首次报道支原体污染对 HEK-293 细胞质粒DNA 转染效率的影响, 并揭 示其机理。

方 法

采用4′,6-二脒基-2-苯基吲哚(DAPI)和聚合酶 链反应(PCR)方法鉴定HEK-293 细胞中的支原 体污染情况以及支原体抗生素Plasmocin 对支原 体污染的清除效果。通过聚乙烯亚胺(PEI)方 法对支原体污染的HEK-293 细胞及支原体清除 后的HEK-293 细胞转染质粒, 比较转染效率差 异。通过高效液相色谱法(HPLC)分析支原体 污染的和支原体清除后的HEK-293 细胞的细胞 裂解产物和细胞上清中的L-精氨酸、瓜氨酸含量 变化。在支原体污染的HEK-293 细胞中补充L-精氨酸, 观察质粒转染效率的改变情况。

结 论

支原体污染能大大降低HEK-293细胞中质粒DNA 的转染效率, 且其原因与支原体能耗竭细胞中的 L-精氨酸有关。


质粒 转染效率 精氨酸 HEK-293 细胞 


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Zi-fei YIN, Ya-ni ZHANG, Juan DU, and Bin-bin CHENG designed this study. Zi-fei YIN and Ya-ni ZHANG performed the experiments. Shu-fang LIANG and Sha-sha ZHAO contributed to data analysis. Zi-fei YIN wrote the manuscript, while Juan DU and Bin-bin CHENG edited the manuscript. All authors read and approved the final manuscript. Therefore, all authors had full access to all the data in the study and take responsibility for the integrity and security of the data.

Compliance with ethics guidelines

Zi-fei YIN, Ya-ni ZHANG, Shu-fang LIANG, Sha-sha ZHAO, Juan DU, and Bin-bin CHENG declare that they have no conflict of interest.

This article does not contain any studies with human or animal subjects performed by any of the authors.

Supplementary material

11585_2019_391_MOESM1_ESM.pdf (175 kb)
Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via l-arginine deprivation in HEK-293 cells


  1. Boslett B, Nag S, Resnick A, 2014. Detection and antibiotic treatment of Mycoplasma arginini contamination in a mouse epithelial cell line restore normal cell physiology. Biomed Res Int 2014: 532105. CrossRefGoogle Scholar
  2. Capiaumont J, Legrand C, Dousset B, et al., 1995. Arginine consumption as a monitor of mycoplasma infection of cultured cells. In Vitro Cell Dev Biol Anim 31(7): 497–498. CrossRefGoogle Scholar
  3. Chernov VM, Chernova OA, Sanchez-Vega JT, et al., 2014. Mycoplasma contamination of cell cultures: vesicular traffic in bacteria and control over infectious agents. Acta Naturae 6(3): 41–51. CrossRefGoogle Scholar
  4. Gedye C, Cardwell T, Dimopoulos N, et al., 2016. Mycoplasma infection alters cancer stem cell properties in vitro. Stem Cell Rev 12(1): 156–161. CrossRefGoogle Scholar
  5. Kim BC, Kim SY, Kwon YD, et al., 2015. Mycoplasma detection and elimination are necessary for the application of stem cell from human dental apical papilla to tissue engineering and regenerative medicine. Biomater Res 19: 6. CrossRefGoogle Scholar
  6. Lin B, Peng GQ, Feng HP, et al., 2017. Purification and characterization of a bioactive alpha-fetoprotein produced by HEK-293 cells. Protein Expr Purif 136: 1–6. CrossRefGoogle Scholar
  7. Riess C, Shokraie F, Classen CF, et al., 2018. Argininedepleting enzymes—an increasingly recognized treatment strategy for therapy-refractory malignancies. Cell Physiol Biochem 51(2): 854–870. CrossRefGoogle Scholar
  8. Strobel B, Zuckschwerdt K, Zimmermann G, et al., 2019. Standardized, scalable, and timely flexible adeno-associated virus vector production using frozen high-density HEK-293 cell stocks and CELLdiscs. Hum Gene Ther Methods 30(1): 23–33. CrossRefGoogle Scholar
  9. Uphoff CC, Drexler HG, 2014. Eradication of mycoplasma contaminations from cell cultures. Curr Protoc Mol Biol 106: 28.5.1-28.5.12. PubMedGoogle Scholar
  10. Uphoff CC, Denkmann SA, Drexler HG, 2012. Treatment of mycoplasma contamination in cell cultures with Plasmocin. J Biomed Biotechnol 2012: 267678. CrossRefGoogle Scholar
  11. Wang LN, Wang Y, Lu Y, et al., 2014. Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo. J Integr Med 12(1): 20–34. CrossRefGoogle Scholar
  12. Zhang YH, Wang Y, Yusufali AH, et al., 2014. Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo. J Integr Med 12(6): 483–494. CrossRefGoogle Scholar

Copyright information

© Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Traditional Chinese Medicine, Changhai HospitalSecond Military Medical UniversityShanghaiChina
  2. 2.Graduate School of Shanghai University of Traditional Chinese MedicineShanghaiChina

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