Construction and identification of a HEK293 cell line with stable TrxR1 overexpression / 南方医科大学学报
Journal of Southern Medical University
;
(12): 554-560, 2022.
Artigo
em Chinês
| WPRIM
| ID: wpr-936347
ABSTRACT
OBJECTIVE@#To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.@*METHODS@#TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.@*RESULTS@#TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).@*CONCLUSION@#We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
RNA Mensageiro
/
Transfecção
/
Auranofina
/
Lentivirus
/
Linhagem Celular Tumoral
/
Células HEK293
/
Vetores Genéticos
Tipo de estudo:
Estudo diagnóstico
Limite:
Humanos
Idioma:
Chinês
Revista:
Journal of Southern Medical University
Ano de publicação:
2022
Tipo de documento:
Artigo
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