Full Text:   <1645>

Summary:  <1132>

Suppl. Mater.: 

CLC number: R34

On-line Access: 2019-11-21

Received: 2019-07-09

Revision Accepted: 2019-09-15

Crosschecked: 2019-10-08

Cited: 0

Clicked: 1889

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Bin-Bin Cheng

https://orcid.org/0000-0002-8321-130X

-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2019 Vol.20 No.12 P.1021-1026

http://doi.org/10.1631/jzus.B1900380


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


Author(s):  Zi-Fei Yin, Ya-Ni Zhang, Shu-Fang Liang, Sha-Sha Zhao, Juan Du, Bin-Bin Cheng

Affiliation(s):  Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; more

Corresponding email(s):   cbb8202@smmu.edu.cn

Key Words:  Mycoplasma, Contamination, Transfection efficiency, HEK-293 cell, Cell culture


Share this article to: More <<< Previous Article|

Zi-Fei Yin, Ya-Ni Zhang, Shu-Fang Liang, Sha-Sha Zhao, Juan Du, Bin-Bin Cheng. Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via L-arginine deprivation in HEK-293 cells[J]. Journal of Zhejiang University Science B, 2019, 20(12): 1021-1026.

@article{title="Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via L-arginine deprivation in HEK-293 cells",
author="Zi-Fei Yin, Ya-Ni Zhang, Shu-Fang Liang, Sha-Sha Zhao, Juan Du, Bin-Bin Cheng",
journal="Journal of Zhejiang University Science B",
volume="20",
number="12",
pages="1021-1026",
year="2019",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1900380"
}

%0 Journal Article
%T Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via L-arginine deprivation in HEK-293 cells
%A Zi-Fei Yin
%A Ya-Ni Zhang
%A Shu-Fang Liang
%A Sha-Sha Zhao
%A Juan Du
%A Bin-Bin Cheng
%J Journal of Zhejiang University SCIENCE B
%V 20
%N 12
%P 1021-1026
%@ 1673-1581
%D 2019
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1900380

TY - JOUR
T1 - Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via L-arginine deprivation in HEK-293 cells
A1 - Zi-Fei Yin
A1 - Ya-Ni Zhang
A1 - Shu-Fang Liang
A1 - Sha-Sha Zhao
A1 - Juan Du
A1 - Bin-Bin Cheng
J0 - Journal of Zhejiang University Science B
VL - 20
IS - 12
SP - 1021
EP - 1026
%@ 1673-1581
Y1 - 2019
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1900380


Abstract: 
mycoplasma infection is the most prevalent contamination in cell culture. Analysis of cell culture in laboratories from different countries shows that mycoplasma contamination ranges from 15% to 80% and, in some cases, even reaches 100% (Chernov et al., 2014). Whilst mycoplasma infection is not visible to the naked eye in cell culture, the consequences of mycoplasma contamination have been shown to induce a number of cellular changes, for example, increased resistance to chemotherapeutic drugs. Therefore, any results obtained from tissue culture studies, in the presence of mycoplasma contamination, potentially render the data invalid (Kim et al., 2015; Gedye et al., 2016). As such, mycoplasmas are not harmless bystanders and cannot be ignored in in vitro studies.

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.
创新点: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.

关键词:支原体;质粒;转染效率;精氨酸;HEK-293细胞

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[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.

[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.

[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.

[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.

[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.

[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.

[7]Riess C, Shokraie F, Classen CF, et al., 2018. Arginine-depleting enzymes—an increasingly recognized treatment strategy for therapy-refractory malignancies. Cell Physiol Biochem, 51(2):854-870.

[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.

[9]Uphoff CC, Drexler HG, 2014. Eradication of mycoplasma contaminations from cell cultures. Curr Protoc Mol Biol, 106:28.5.1-28.5.12.

[10]Uphoff CC, Denkmann SA, Drexler HG, 2012. Treatment of mycoplasma contamination in cell cultures with Plasmocin. J Biomed Biotechnol, 2012:267678.

[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.

[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.

Open peer comments: Debate/Discuss/Question/Opinion

<1>

Please provide your name, email address and a comment





Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2022 Journal of Zhejiang University-SCIENCE