Construction of Stable Cell Lines with TOE1 Knockdown and Overexpression and Analysis on its Cellular Localization in Gastric Cancer Cells

doi:10.19586/j.2095-2341.2022.0197

Current Biotechnology ›› 2023, Vol. 13 ›› Issue (2): 273-281.DOI: 10.19586/j.2095-2341.2022.0197

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Construction of Stable Cell Lines with TOE1 Knockdown and Overexpression and Analysis on its Cellular Localization in Gastric Cancer Cells

Xiaolin SUN(), Didi ZHANG, Xinya WANG, Lina ZHANG()

Faculty of Environment and Life，Beijing University of Technology，Beijing 100124，China

Received:2022-11-16 Accepted:2022-12-16 Online:2023-03-25 Published:2023-04-07
Contact: Lina ZHANG

Abstract

Abstract:

TOE1 （target of Egr1） is a newly discovered isoenzyme of DEDD superfamily deadenylase Caf1. Construction of its knockdown and overexpression stable cell lines， and analysis of its cellular localization can provide a basis for further research on the function of TOE1 gene. Firstly， the shRNA sequence targeting the silencing of TOE1 gene was inserted into the lentiviral vector pLVX-shRNA2-Puro to construct the lentiviral vector pLVX-shTOE1 that knocks down TOE1 gene. Then， the TOE1 gene sequence was amplified by PCR and cloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro to construct the lentiviral vector pCDH-TOE1 with overexpression of TOE1 gene. Meanwhile， the eukaryotic expression vector pEGFP-TOE1 was constructed by the same method and transfected into human gastric cancer cell SGC-7901， and the subcellular localization of TOE1 was observed by confocal microscopy. After lentivirus packaging， SGC-7901 cells were infected with pLVX-shTOE1 and pCDH-TOE1 lentivirus， and then SGC-7901-shTOE1 cell line stably silenced TOE1 gene and SGC-7901-oeTOE1 cell line stably overexpressed TOE1 gene were selected by puromycin. Next， the knockdown and overexpression efficiencies of TOE1 at mRNA and protein levels were verified by RT-qPCR and Western blot， respectively. The identification results showed that the expression of TOE1 in SGC-7901-shTOE1 cells was significantly decreased， and the expression of TOE1 in SGC-7901-oeTOE1 cells was obviously increased， suggesting that TOE1-silenced and TOE1-overexpressed SGC-7901 stable gastric cancer cell lines were successfully constructed. Also， the confocal results showed that TOE1 was localized in the nucleus. The results laied a foundation for further exploring the function and mechanism of TOE1 in gastric cancer.

Key words: TOE1, stable cell line, lentiviral vector, SGC-7901 cells, cellular localization

CLC Number:

Q279

Xiaolin SUN, Didi ZHANG, Xinya WANG, Lina ZHANG. Construction of Stable Cell Lines with TOE1 Knockdown and Overexpression and Analysis on its Cellular Localization in Gastric Cancer Cells[J]. Current Biotechnology, 2023, 13(2): 273-281.

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URL: https://swjsjz.magtechjournal.com/EN/10.19586/j.2095-2341.2022.0197

https://swjsjz.magtechjournal.com/EN/Y2023/V13/I2/273

Figures/Tables 7

Fig. 1 Schematic diagram of pLVX-shTOE1 and pCDH-TOE1 lentiviral vector construction

Fig. 2 Sequencing comparison results of pLVX-shTOE1 lentiviral vector

Fig. 3 PCR amplification, electrophoretic identification and sequencing comparison results of TOE1 gene

Fig. 4 Fluorescence results of SGC-7901 stable cell line with TOE1 gene knockdown (×10)

Fig. 5 Expression of TOE1 mRNA in gastric cancer stable cell lines

Fig. 6 Expression of TOE1 protein in gastric cancer stable cell lines

Fig. 7 Subcellular localization of TOE1 in SGC-7901 cells (×100)

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Construction of Stable Cell Lines with TOE1 Knockdown and Overexpression and Analysis on its Cellular Localization in Gastric Cancer Cells

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