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To study function of some gene, the most commonly used method is to overexpress the gene or knock-down the gene through RNA interference in the host cell, and the routine ways include transient transfection and selection of stable cell line.
The selected overexpression or RNAi stable cell lines of target gene can bring great convenience to your experiments. With stable cell line, later Co-IP or Pull-Down experiments will be very handy; cell line with stable expression of recombinant fluorescent protein allows you to dynamically observe the movement of molecules in the cell. The basic principle to establish stable cell line is to clone exogenous DNA into vector of some resistance, then to transfect vector into host cell and integrate it into chromosome, and to screen cells using resistance markers contained in vector. The most commonly used resistance selection markers of eukaryotic expression vector include neomycin, hygromycin and puromycin. Usually G418 is used to replace neomycin for selective screening. After selection cell lines with stable expression of target protein or silenced specific gene can be obtained.
1、After transfection of plasmid, screen stable cell lines by method of monoclone;
2、Screen stable cell lines by lentivirus infection.
Compared with plasmid transfection and monoclone screening, it is more convenient and efficient to use lentivirus infection for selection, which is currently the primary method to select stable cell line. Lentivirus can infect almost all kinds of cells, and can be integrated into genome of Host cells after infection for long-term stable expression. Thus, lentivirus is often used for preparation of monoclonal cell lines for stable expression/silence of specific gene.
Lentivirus constructed and packed by HANBIO has resistance to puromycin, which can be selectively screened by adding puromycin after Host cells infection, to obtain cell lines for stable expression of shRNA or specific gene.We offer Lentivirus packing services (you can do the stable cell line selection by yourself), or one-stop screen stable cell lines service.
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