Construction and Validation of Lentivirus-mediated Stable Knockdown of SKM-1 in <i>TET2</i> Gene

doi:10.13523/j.cb.2310047

China Biotechnology

2023, Vol. 43

Issue (12): 160-168 DOI: 10.13523/j.cb.2310047

Construction and Validation of Lentivirus-mediated Stable Knockdown of SKM-1 in TET2 Gene

GU Xiao-li,YANG Xiu-peng,YU Li,LING Zhi-ming,XU Yong-gang(

)

Department of Hematology, Xiyuan Hospital, China Academy of Traditional Chinese Medicine, Beijing 100091, China

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Abstract

Objective: To construct the SKM-1 cell line with stable knockdown of the TET2 gene, and to infect SKM-1 cell line with the packaged H_TET2-shRNA lentivirus to obtain the H_TET2-shRNA SKM-1 stable strain. Methods: The SKM-1 cell line with stable knockdown of TET2 gene was verified by qPCR and Western blot (WB) experiments. The proliferative activity and apoptosis of SKM-1 cells before and after transfection were detected using the cell counting kit-8 (CCK-8) cell viability assay and flow cytometry, respectively. Results: qPCR verified that the TET2 gene expression was significantly reduced in SKM-1 cells with H_TET2-shRNA. Western blot showed that the TET2 protein expression was also significantly reduced in SKM-1 cells with H_TET2-shRNA and the CCK-8 cell viability assay confirmed that stable knockdown of the TET2 gene did not affect cell viability. The cell cycle assay revealed that S-phase cells accounted for 48.1% of the total cell number after knockdown of TET2, which was higher than that of the SKM-1 group (42.3%) and the control empty plasmid group. The apoptosis assay revealed that the apoptosis rate of SKM-1 cells in the TET2 knockdown group was 0.6% lower than that of the empty plasmid group (4.4%). Conclusions: SKM-1 cell line with stable low expression of the TET2 gene was constructed by lentivirus-mediated transfection, and the constructed cell line was verified to be successful by using qPCR and WB experiments. It was confirmed that the stable knockdown of TET2 did not affect the viability of the cells by using the CCK-8 cell viability assay. The cell cycle assay of flow cytometry found that the knockdown of TET2 affected the function of cellular DNA replication, which led to the abnormalities of cellular interphase division and affected cell growth and self-repair. The flow cytometry apoptosis assay found that knockdown of TET2 would inhibit apoptosis, which may be related to the fact that TET2 is an anti-oncogene.

Key words： TET2 knockdown cell line SKM-1 cell line Lentiviral packaging Cell cycle Apoptosis

Received: 18 September 2023 Published: 16 January 2024

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Xiao-li GU, Xiu-peng YANG, Li YU, Zhi-ming LING, Yong-gang XU. Construction and Validation of Lentivirus-mediated Stable Knockdown of SKM-1 in TET2 Gene. China Biotechnology, 2023, 43(12): 160-168.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2310047 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I12/160

Fig.1 Results of the TET2 qPCR assay ns:P>0.05;^* :P≤0.05;^** :P≤0.01;^*** :P≤0.001;^**** :P≤0.000 1

Fig.2 Carrier information

Table 1 sequencing results of shRNA interference vectors

Table 2 Titer scale

Fig.3 Results of H_TET2-shRNA qPCR detection ns:P>0.05;^* :P≤0.05;^** :P≤0.01;^*** :P≤0.001;^**** :P≤0.000 1

Fig.4 Results of H_TET2-shRNA Western blot detection S1:Control group; S2:Empty plasmid group; S3:H_TET2-shRNA215; S4:H_TET2-shRNA576; Primary antibody: 1∶1 000; Secondary antibody: 1∶3 000

Fig.5 Effect of H_TET2-shRNA on SKM-1 cell cycle as measured by flow cytometry A. Cell OD values at various time points B. Growth trends of various cell lines over time C. H_TET2-shRNA group D. SKM-1 group E. NC group. ns:P>0.05;^* :P≤0.05;^** :P≤0.01;^*** :P≤0.001;^**** :P≤0.000 1

Fig.6 Effect of H_TET2-shRNA on SKM-1 cell apoptosis as measured by flow cytometry A.Empty plasmid nude cell group B. H_TET2-shRNA naked cell group C. Empty plasmid group D.H_TET2-shRNA group


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