Establishment and Functional Analysis of MDCK Cell Line Induced IFITM3 Expression Based on Tet-On 3G System†

doi:10.7503/cjcu20160794

Chem. J. Chinese Universities ›› 2017, Vol. 38 ›› Issue (5): 770.doi: 10.7503/cjcu20160794

• Organic Chemistry • Previous Articles Next Articles

Establishment and Functional Analysis of MDCK Cell Line Induced IFITM3 Expression Based on Tet-On 3G System^†

CAO Tingting^1,², DU Shouwen¹, XU Wang¹, XING Bin¹, ZHAO Fei¹, WANG Maopeng¹, ZHU Yilong¹, BAI Jieying¹, TIAN Yufei¹, LIU Liming³, ZHAO Cuiqing³, ZHOU Yifa², LI Chang^1,^3,^4,^*(), JIN Ningyi^1,^3,^4,^*()

1. Military Veterinary Institute, Academy of Military Medical Sciences, Key Labtoratory of Jinlin Province for Zoonosis Prevention and Control, Changchun 130122, China
2. College of Life Sciences, Northeast Normal University, Changchun 130022, China
3. Institute of Virology, Wenzhou University, Wenzhou 325035, China
4. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China

Received:2016-11-16 Online:2017-05-10 Published:2017-04-20
Contact: LI Chang,JIN Ningyi E-mail:lichang78@163.com;ningyik@126.com
Supported by:
† Supported by the National Natural Science Foundation of China(Nos.31472197, 31402175), the Open Project of State Key Laboratory of Pathogen and Biosecurity, China(No.SKLPBS1435), the Beijing Natural Science Foundation, China(No.5152023) and the Youth Foundation of Jilin Province, China(No.20140520173JH)

Abstract

Abstract:

To explore the antiviral mechanisms of IFITM3, the eukaryotic expression plasmid pTRE3G-IFITM3 containing IFITM3 gene was constructed based on Tet-On 3G system and then cotranfected with the regulator vector pLVX-Tet3G into MDCK cells. After 48 h, the cells were subjected to select with G418 and puromycin, followed by treatment with or without doxycycline(Dox) to identify IFITM3 expression, continuing to Dox sensitivity analysis, IFITM3⁺ cell percentage and location analysis. And then, infection by lentiviruses pseudotyped with avian influence virus H5 or H7 hemagglutinin or VSV G proteins was performed to detect luciferase activities. The results indicated that inducible IFITM3-expressing MDCK cell lines were obtained and expression level of IFITM3 was correlated with the dose and induction time of Dox. The concentration of Dox was determined to be 2.5 g/mL, and IFITM3⁺ cell percentage was up to 75% or more after 12 h. And IFITM3 was distributed in late endosomes/lysosomes and efficiently suppressed the avian influence virus H5 or H7 hemagglutinin or VSV G-mediated viral entry, to lay a foundation for further research into the inhibition mechanism.

Key words: Interferon-inducible transmembrane protein 3, Interferon-stimulated genes, Inducible expression, Virus entry, Restriction

CLC Number:

TrendMD:

CAO Tingting, DU Shouwen, XU Wang, XING Bin, ZHAO Fei, WANG Maopeng, ZHU Yilong, BAI Jieying, TIAN Yufei, LIU Liming, ZHAO Cuiqing, ZHOU Yifa, LI Chang, JIN Ningyi. Establishment and Functional Analysis of MDCK Cell Line Induced IFITM3 Expression Based on Tet-On 3G System^†[J]. Chem. J. Chinese Universities, 2017, 38(5): 770.

Figures/Tables 7

Fig.1 Construction and identification of recombinant plasmid pTRE3G-IFITM3(B)

Fig.2 Identification of IFITM3 expression induced by Dox when cells were co-transfected with pTRE3G-IFITM3 and pLVX-Tet3G

Fig.3 Identification of MDCK cell line induced IFITM3 expression based on Tet-On 3G system by RT-PCR(A) and Western blot(B) in transcriptional and translational levels

Fig.4 Correlation between IFITM3 expression level with the concentrations and induction time of doxycycline(Dox)

Fig.5 Effect of doxycycline on cell viability analyzed by MTS at the different time points following the treatment with Dox or DMSO

Fig.6 IFITM3+ cell percentage(A) and distribution(B) following induced with Dox monitored through flow cytometry or immunofluorescence imaging technology(200×)(A) * P<0.001 vs. DMSO; (B) IFITM3 was labeled with FITC fluorescent probe, which excitation wavelength is 488 nm;LBPA was labeled with Cy3 probe, which excitation wavelength is 570 nm.

Fig.7 IFITM3-mediated suppressions to influenza A virus hemagglutinin(HA) or vesicular stomatitis virus glycoprotein(VSVG)-mediated viral entry analyzed by luciferase reporter system

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Establishment and Functional Analysis of MDCK Cell Line Induced IFITM3 Expression Based on Tet-On 3G System^†

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