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* These authors contributed equally
This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4α, RXRα and PPARα) and traditional hepatic cells (HepG2-NE, expressing human NTCP).
HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T overexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines. HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.
Hepatitis B affects the lives of more than 2 billion people and is one of the major threats to public health. Approximately 257 million people are chronically infected with hepatitis B virus (HBV) worldwide, causing a big burden to the society1. Hepatocytes are not the only cells infected by HBV and other cells in non-hepatic tissue, such as kidney and testis, are also infected by this virus2,3. Currently, HBV infection cell models are limited to human hepatocytes with only few non-hepatic cell line models. This hampers the study of HBV infection and HBV-related pathology of non-hepatic....
Culture, collection of supernatants from HepG2.2.15 cells and HBV infection should be performed in biosafety level II (P2) or biosafety level III (P3) laboratory according to the biosafety guidance in the country. Laboratory safety practices should be followed to ensure the safety of laboratory personnel, and all should be vaccinated and detected HBsAb positive before performing HBV experiments. Observe the state of the cells at every step before proceeding to the next step. Human serum samples were used in accordance wi.......
We constructed pSIN-NTCP-EGFP plasmid expressing NTCP and EGFP fusion and with puromycin resistance. The plasmid was transfected into HepG2 and 293T cells to construct stable cell lines HepG2-NE and 293T-NE expressing NTCP and EGFP. Plasmids (pSIN-HNF4α, pSIN-RXRα, pLV-PPARα-puro-flag) with puromycin resistance and expression were transfected into 293T-NE cells to construct a stable cell line expressing 4 host genes9. The expression of NTCP-EGFP can be observed by green fluorescence.......
Here, we introduce protocols for HBV infection in non-hepatic 293T-NE-3NRs and hepatoma-based HepG2-NE cells. 293T-NE-3NRs were suitable for HBV infection at both high and low GEq. The following critical steps need to be taken into consideration while using our protocol. The cell status is an important factor for a successful infection. The infection medium must be changed timely after the initial period of HBV infection. 293T-NE-3NRs cells are typically fragile following infection with high viral titers. Therefore, thes.......
This work was supported by the National Natural Science Foundation of China (No. 81870432 and 81570567 to X.L.Z.), (No. 81571994 to P.N.S.), Research Fund for International Young Scientists (No. 81950410640 to W.I.); The Li Ka Shing Shantou University Foundation (No. L1111 2008 to P.N.S.). We would like to thank Prof. Stanley Lin from Shantou University Medical College for useful advice.
....Name | Company | Catalog Number | Comments |
0.45μm membrane filter | Millex-HV | SLHU033RB | Filter for HepG 2.2.15 supernatant |
293T-NE | Laboratory construction | —— | Cell model for HBV infection |
293T-NE-3NRs | Laboratory construction | —— | Cell model for HBV infection |
594 labeled goat against rabbit IgG | ZSGB-BIO | ZF-0516 | For immunofluorescence assay,second antibody |
6-well plate | BIOFIL | TCP010006 | For co-culture |
Amicon Ultra 15 ml | Millipore | UFC910008 | For concentration of HepG 2.2.15 supernatant |
BSA | Beyotime | ST023 | For immunofluorescence assay |
Cyclosporin A | Sangon biotech | 59865-13-3 | inhibitor of HBV infection |
DAPI | Beyotime | C1006 | For nuclear staining |
Diagnostic kit for Quantification of Hepatitis B Virus DNA(PCR-Fluorescence Probing) | DAAN GENE | 7265-2013 | For HBV DNA detection |
DMEM | HyClong | SH30243.01 | For culture medium |
DMSO | Sigma-Aldrich | D5879 | For improvement of infection efficiency |
Fetal bovine serum(FBS) | CLARK Bioscience | FB25015 | For culture medium |
Fluorescence microscope | ZEISS | Axio observer Z1 | For immunofluorescence assay |
HepG2-NE | Laboratory construction | —— | Cell model for HBV infection |
HBcAg antibody | ZSGB-BIO | ZA-0121 | For immunofluorescence assay, primary antibody |
PBS | ZSGB-BIO | ZLI-9062 | For cell wash |
PEG8000 | Merck | P8260 | For infection medium |
Penicillin-Streptomycin-Glutamine | Thermo Fisher | 10378016 | For culture medium |
Transwell | CORNING | 3412 | For co-culture |
Tween 20 | sigma-Aldrich | WXBB7485V | For PBST |
Virkon | Douban | 6971728840012 | Viruside |
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