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Establishment of stable cell lines in which the HBV genome replicates episomally for evaluation of antivirals.

ABSTRACT: Introduction:Due to the increasing resistance to nucleot(s)ide analogs in patients with chronic hepatitis B, development of new antiviral drugs to eradicate hepatitis B virus is still urgently needed. Material and methods:To date, most studies on evaluating anti-HBV drugs have been performed using cell lines where the HBV genomic DNA is chromosomally integrated, e.g. Hep2.2.15 in HBV-infected livers of the viral episomal genome replicates in the nucleus and covalently closed circular DNA (cccDNA) serves as a transcriptional template. Another option involves the use of HBV-infected cells of HepaRG or NTCP-overexpressing cells. However, the development of the infection system is expensive and laborious, and its HBV expression level remained low. Results:Compared to HuH7 cells, the established stable cell lines based on episomal-type pEB-Multi vectors can been expressed HBV wild-type by qRT-PCR and immunoblotting (p < 0.05). These two vectors are also sensitive to Entecavir and against nucleoside analog Lamivudine in mutants cellines. Conclusions:It is worth demonstrating how useful the established cell system is for evaluating antiviral agents and their mechanisms of action.

SUBMITTER: Sun S

PROVIDER: S-EPMC7069427 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Establishment of stable cell lines in which the HBV genome replicates episomally for evaluation of antivirals.

Sun Suofeng S Li Yuan Y Liu Bowei B Zhang Bingyong B Han Shuangyin S Li Xiuling X

Archives of medical science : AMS 20181120 2

<h4>Introduction</h4>Due to the increasing resistance to nucleot(s)ide analogs in patients with chronic hepatitis B, development of new antiviral drugs to eradicate hepatitis B virus is still urgently needed.<h4>Material and methods</h4>To date, most studies on evaluating anti-HBV drugs have been performed using cell lines where the HBV genomic DNA is chromosomally integrated, e.g. Hep2.2.15 in HBV-infected livers of the viral episomal genome replicates in the nucleus and covalently closed circu ...[more]

PMID: 32190152

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Project description:Background:Canine prostate cancer represents a unique model for human prostate cancer. In vitro systems offer various possibilities but Xenograft in vivo imaging allows studying complex tasks as tumor progression and drug intervention longitudinal. Herein, we established three canine prostate carcinoma cell lines stably expressing fluorescent proteins allowing deep tissue in vivo imaging. Methods:Three canine prostate carcinoma (cPC) cell lines were stably transfected with fluorescent proteins in red, far-red and near infra-red spectrum, followed by G418 selection. Fluorescent protein expression was demonstrated by microscopy, flow cytometry and a NightOWL LB 983 in vivo imaging system. Cellular and molecular characteristics of the generated cell lines were compared to the parental cell line CT1258. Cell proliferation, metabolic activity and sphere formation capacity were analyzed. Stem cell marker expression was examined by qPCR and genomic copy number variation by genomic DNA whole genome sequencing. Results:Three stably fluorescent protein transfected cPC cell lines were established and characterized. Compared to the parental cell line, no significant difference in cell proliferation and metabolic activity were detected. Genomic copy number variation analyses and stem cell marker gene expression revealed in general no significant changes. However, the generated cell line CT1258-mKate2C showed uniquely no distal CFA16 deletion and an elevated metabolic activity. The introduced fluorescencent proteins allowed highly sensitive detection in an in vivo imaging system starting at cell numbers of 0.156?×?106. Furthermore, we demonstrated a similar sphere formation capacity in the fluorescent cell lines. Interestingly, the clone selected CT1258-mKate2C, showed increased sphere formation ability. Discussion:Starting from a well characterized cPC cell line three novel fluorescent cell lines were established showing high cellular and molecular similarity to the parental cell line. The introduction of the fluorescent proteins did not alter the established cell lines significantly. The red fluorescence allows deep tissue imaging, which conventional GFP labeling is not able to realize. Conclusion:As no significant differences were detected between the established cell lines and the very well characterized parental CT1258 the new fluorescent cell lines allow deep tissue in vivo imaging for perspective in vivo evaluation of novel therapeutic regimens.

Dataset Information

Establishment of stable cell lines in which the HBV genome replicates episomally for evaluation of antivirals.

Publications

Establishment of stable cell lines in which the HBV genome replicates episomally for evaluation of antivirals.

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