一种高效稳定的磷酸钙转染293T细胞方法的建立及评价

doi:10.13481/j.1671-587x.20190535

吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (05): 1177-1181.doi: 10.13481/j.1671-587x.20190535

• 方法学 • 上一篇

一种高效稳定的磷酸钙转染293T细胞方法的建立及评价

华进¹, 程志彬^1,2, 林春霖¹, 戴起宝¹, 朱广伟^1,2

1. 福建医科大学附属第一医院胃肠外科二区, 福建福州 350005;
2. 福建医科大学消化道恶性肿瘤教育部重点实验室, 福建福州 350005

收稿日期:2019-01-19 发布日期:2019-10-08
通讯作者: 朱广伟,主治医师,硕士研究生导师(Tel:0591-87982082,E-mail:zgwzsy@126.com) E-mail:zgwzsy@126.com
作者简介:华进(1981-),男,福建省南平市人,主治医师,医学硕士,主要从事炎症性肠病基础与胃肠外科临床方面的研究。
基金资助:
国家自然科学基金资助课题（81702424，81872364）；国家卫健委临床重点专科建设项目（普通外科）资助课题（2013）；福建省科技厅自然科学基金资助课题（2018J05127）；福建省卫健委中青年骨干项目资助课题（2018-ZQN-46）；福建省教育厅中青年教师教育科研项目资助课题（JAT170239）

Establishment and evaluation of a highly efficient and stable calcium phosphate transfection method for 293T cells

HUA Jin¹, CHENG Zhibin^1,2, LIN Chunlin¹, DAI Qibao¹, ZHU Guangwei^1,2

1. Department of Gastrointestinal Surgery 2 Section, First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China;
2. Key Laboratory of Gastrointestinal Cancer, Ministry of Education, Fujian Medical University, Fuzhou 350005 China

Received:2019-01-19 Published:2019-10-08

摘要/Abstract

摘要： 目的：探讨2种磷酸钙转染法转染293T细胞的效果，建立一种实现高效稳定磷酸钙转染293T细胞的方法。方法：荧光显微镜观察采用2种磷酸钙转染方法（传统磷酸钙转染法和改良磷酸钙转染法）转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞的转染效率。采用Real-time PCR法和Western blotting法分别检测2种磷酸钙转染方法转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞后，肿瘤坏死因子受体相关因子6（TRAF6） mRNA和flag蛋白表达水平。结果：荧光显微镜下观察，与传统磷酸钙转染法比较，改良磷酸钙转染法转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞24和48 h后的转染效率明显升高（P<0.01）；Real-time PCR法和Western blotting法检测，与传统转染方法比较，磷酸钙转染改良法转染pCDH-GFP-3xflag-TRAF6质粒进入293T细胞24和48 h后，TRAF6 mRNA和flag蛋白表达水平均明显升高（P<0.01）。结论：本研究建立的改良磷酸钙转染方法是一种高效、稳定的DNA转染方法。

关键词: 磷酸钙转染, 293T细胞, 肿瘤坏死因子受体相关因子6, flag蛋白

Abstract: Objective:To explore the effects of two kinds of calcium phosphate transfection methods in the 293T cells, and to establish the method of achieving high-efficiency and stable calcium phosphate transfection in the 293T cells. Methods:Fluorescence microscope was used to observe the transfection efficiencies of transfection of pCDH-GFP-3xflag-TRAF6 plasmid into the 293T cells by two kinds of calcium phosphate transfection methods (traditional calcium phosphate transfection method and improved calcium phosphate transfection method). Real-time PCR and Western blotting method were used to detect the expression levels of TRAF6 mRNA and flag protein in the 293T cells after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by two kinds of calcium phosphate transfection methods. Results:Under fluorescence microscope, compared with traditional calcium phosphate transfection method, the transfection efficiencies of improved calcium phosphate transfection method 24 an 48 h after transfection of pCDH-GFP-3xflag-TRAF6 plasmid into the 293T cells were significantly increased (P<0.01). The Real-time PCR and Western blotting results showed that compared with traditional transfection method, the expression levels of TRAF6 mRNA and flag protein in the 293T cells 24 and 48 h after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by calcicum phosphate transfection method were significantly increased(P<0.01). Conclusion:The improved calcium phosphate transfection method established in this reseach is a highly efficient and stable DNA transfection method.

Key words: calcium phosphate transfection, 293T cells, tumor necrosis factor receptor associated factor 6, flag protein

中图分类号:

Q819

华进, 程志彬, 林春霖, 戴起宝, 朱广伟. 一种高效稳定的磷酸钙转染293T细胞方法的建立及评价[J]. 吉林大学学报(医学版), 2019, 45(05): 1177-1181.

HUA Jin, CHENG Zhibin, LIN Chunlin, DAI Qibao, ZHU Guangwei. Establishment and evaluation of a highly efficient and stable calcium phosphate transfection method for 293T cells[J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1177-1181.

参考文献

[1] RECILLAS T F. Multiple strategies for gene transfer, expression, knockdown, and chromatin influence in mammalian cell lines and transgenic animals[J]. Mol Biotechnol, 2006, 34(3):337-354.
[2] 唐蓓. 一种稳定且高效的磷酸钙293T细胞转染法[J]. 生物技术, 2011, 21(1):49-51.
[3] GRAHAM F L, VAN DER E B A J. A new technique for the assay of infectivity of human adenovirus 5 DNA[J]. Virology, 1973, 52(2):456-467.
[4] ZHAO D, WANG C Q, ZHUO R X, et al. Modification of nanostructured calcium carbonate for efficient gene delivery[J]. Colloid Surface B, 2014, 118:111-116.
[5] CHENUET S, MARTINET D, BESUCHET S N, et al. Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection[J]. Biotechnol Bioeng, 2008, 101(5):937-945.
[6] ALAM J, COOK J L. Reporter genes:application to the study of mammalian gene transcription[J]. Anal Biochem, 1990, 188(2):245-254.
[7] KWON M, FIRESTEIN B L. DNA transfection:calcium phosphate method[J]. Methods Mol Biol, 2013, 1018:107-110.
[8] DUDEK H, GHOSH A, GREENBERG M E. Calcium phosphate transfection of DNA into neurons in primary culture[J]. Curr Protoc Neurosci, 1998,3(1):3.11.1-3.11.6.
[9] GUO L, WANG L, YANG R, et al. Optimizing conditions for calcium phosphate mediated transient transfection[J]. Saudi J Biol Sci, 2017, 24(3):622-629.
[10] SUN M, BERNARD L P, DIBONA V L, et al. Calcium phosphate transfection of primary hippocampal neurons[J]. J Vis Exp, 2013(81):e50808.
[11] INOH Y, MIE N G, MATSUSHITA K, et al. Gene transfection efficiency into dendritic cells is influenced by the size of cationic liposomes/DNA complexes[J]. Eur J Pharm Sci, 2017, 102:230-236.
[12] VILLA-DIAZ L G, GARCIA PEREZ J L, KREBSBACH P H. Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix[J]. Stem Cells Dev, 2010, 19(12):1949-1957.
[13] SWIFT S, LORENS J, ACHACOSO P. Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems[J]. Curr Protoc Immunol, 1999:10.17.14-10.17.29.
[14] MAO Y Y, YAN R H, LI A, et al. Lentiviral vectors mediate long-term and high efficiency transgene expression in HEK 293T cells[J]. Int J Med Sci, 2015, 12(5):407-415.
[15] ALBRECHT C, HOSINER S, TICHY B, et al. Comparison of lentiviral packaging mixes and producer cell lines for RNAi applications[J]. Mol Biotechnol, 2015, 57(6):499-505.
[16] RIPPE A, BRENNER A, LEFFERT L. DNA-mediated gene transfer into adult rat hepatocytes in primary culture[J]. Mol Cell Biol, 1990, 10(2):689-695.
[17] ZHOU Q, WANG Y, XIANG J J, et al. Stabilized calcium phosphate hybrid nanocomposite using a benzoxaborole-containing polymer for pH-responsive siRNA delivery[J]. Biomater Sci, 2018, 6(12):3178-3188.

一种高效稳定的磷酸钙转染293T细胞方法的建立及评价

Establishment and evaluation of a highly efficient and stable calcium phosphate transfection method for 293T cells

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