Construction of a tetracycline-induced expression system for recombinant protein expression in large yellow croaker (Larimichthys crocea) cells

doi:10.16178/j.issn.0528-9017.20231217

Abstract

Abstract:

In order to construct a tetracycline-induced expression system for recombinant protein expression in large yellow croaker (Larimichthys crocea) cells to study the protein function of large yellow croaker, a Tet-On system recombinant plasmid, designated as pTRE3G-CMV, was constructed by replacing the EF-1α promoter and its downstream 5'-long terminal repeat (5'-LTR) sequence of transcription activator (rtTA) of a Tet-On 3G inducible expression plasmid pTRE3G-MCS-EGFP-3×FLAG-Tetone-Puro with the CMV enhancer/promoter of pEGFP-N1 plasmid. When transfected YCK-hk (large yellow croaker head kidney cell line) cells with pTRE3G-CMV plasmid DNA by lipofection reagent, the expression of EGFP positive cells was observed post doxycycline induction for 3 h, but the percentages of EGFP positive cells was low (<1%). After screened with purinomycin, the EGFP positive ratio of cells can be increased to about 30%, suggesting that the pTRE3G-CMV stable cell lines can be established by combined with purinomycin screening technique and single-cell clonal lines screening technique. These results indicated that the Tet-On plasmid pTRE3G-CMV constructed in this study can normally express foreign genes in large yellow croaker cells and can be used to study the protein function of large yellow croaker in vitro.

Key words: tetracycline-induced expression system, Larimichthys crocea, recombinant protein expression

CLC Number:

GE Jinxin, YAN Tingding, WANG Kezhi, LIU Ye, HE Zhiqiao, SHEN Wang. Construction of a tetracycline-induced expression system for recombinant protein expression in large yellow croaker (Larimichthys crocea) cells[J]. Journal of Zhejiang Agricultural Sciences, 2024, 65(6): 1285-1290.

Figures/Tables 5

Table 1 Primer information

引物名称	序列(5' → 3')	用途
N1CMVF	GAATCTAAGTGGATCTGCGATCGCTCCGGTGCCCGTCAGTTAGTTATTAATAGTAATCA	载体构建
N1CMVR	TATGACTTTGCTCTTGTCCAGTCTAGACATGGTGGCGGCGAGTCCGGTAGCGCTAGC	载体构建

Fig.1 pTRE3G-CMV carrier construction process

Fig.2 Electrophoretic construction of pTRE3G-CMV carrier

Fig.3 Expression of EGFP by pTRE3G-CMV plasmid in PCK-HK cells induced by Dox (100×)

Fig.4 Screening of pTRE3G-CMV transfected cells with purinomycin (200×)

References 19

[1]	农业农村部渔业渔政管理局,全国水产技术推广总站, 中国水产学会编制. 中国渔业统计年鉴-2022[M]. 北京: 中国农业出版社, 2022: 22.
[2]	游宇, 李水根, 廖碧钗, 等. 福建配合饲料替代幼杂鱼现状分析与对策建议[J]. 中国水产, 2023(11): 48-51.
[3]	邓益琴. 水产动物弧菌病及其生物防治研究进展[J]. 大连海洋大学学报, 2023, 38(4): 553-563.
[4]	蒋英芝, 贺连华, 刘建军. 蛋白质功能研究方法及技术[J]. 生物技术通报, 2009(9): 38-43.
[5]	DAS A T, ZHOU X, METZ S W, et al. Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells[J]. Biotechnology Journal, 2016, 11(1): 71-79.
[6]	汪慧娟, 何志巧, 石戈, 等. 大黄鱼(Larimichthys crocea)tristetraprolin基因的克隆与功能分析[J]. 海洋与湖沼, 2023, 54(6): 1786-1796.
[7]	张宏祥, 李坤明, 王娜, 等. 斑石鲷肝脏细胞系的建立与鉴定[J]. 农业生物技术学报, 2020, 28(2): 360-368.
[8]	王贤慧, 慕鹏飞, 敖敬群. 大黄鱼头肾细胞系瞬时转染方法的选择及优化[J]. 生物技术, 2023, 33(3): 287-292, 321.
[9]	KALLUNKI T, BARISIC M, JÄÄTTELÄ M, et al. How to choose the right inducible gene expression system for mammalian studies?[J]. Cells, 2019, 8(8): 796.
[10]	OTOMO J, WOLTJEN K, SAKURAI H. Uniform transgene activation in Tet-On systems depends on sustained rtTA expression[J]. Science, 2023, 26(10): 107685.
[11]	STIEGER K, BELBELLAA B, LE GUINER C, et al. In vivo gene regulation using tetracycline-regulatable systems[J]. Advanced Drug Delivery Reviews, 2009, 61(7/8): 527-541.
[12]	MUÑOZ I, CARRILLO M, ZANUY S, et al. Regulation of exogenous gene expression in fish cells: an evaluation of different versions of the tetracycline-regulated system[J]. Gene, 2005, 363: 173-182.
[13]	KNOPF F, SCHNABEL K, HAASE C, et al. Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish[J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(46): 19933-19938.
[14]	WEST M C, CAMPBELL L J, WILLOUGHBY J J, et al. Two types of transgenic lines for doxycycline-inducible, cell-specific gene expression in zebrafish ultraviolet cone photoreceptors[J]. Gene Expression Patterns, 2014, 14(2): 96-104.
[15]	WEHNER D, JAHN C, WEIDINGER G. Use of the TetON system to study molecular mechanisms of zebrafish regeneration[J]. Journal of Visualized Experiments, 2015(100): e52756.
[16]	张力, 刘超, 周昕, 等. 四环素诱导肝脏特异表达绿色荧光蛋白转基因斑马鱼模型建立[J]. 中国实验动物学报, 2015, 23(1): 81-85.
[17]	KAYO D, KIMURA S, YAMAZAKI T, et al. Spatio-temporal control of targeted gene expression in combination with CRISPR/Cas and Tet-On systems in Medaka[J]. Genesis, 2023: e23519.
[18]	CUI K, LI Q, XU D, et al. Establishment and characterization of two head kidney macrophage cell lines from large yellow croaker (Larimichthys crocea)[J]. Developmental and Comparative Immunology, 2020, 102:103477.
[19]	WANG X H, LI Q H, MU P F, et al. Large yellow croaker peroxiredoxin Ⅳ protect cells against oxidative damage and apoptosis[J]. Molecular Immunology, 2020, 127: 150-156.