Definition of transfected stable cell lines Stable transfection means integrating plasmid DNA with exogenous gene into the host cell chromosomes, which then express the target gene and protein continuously. After transient transfection, if necessary, with the help of virus with high transfection efficiency, target cells would be screened. Afterwards, cells were passaged by appropriately selective drugs according to the different resistance markers contained in the plasmid vectors, so that transfected stable expressing cell lines can be obtained.
Prophase preparation:
- Construct vectors of foreign gene, target gene, shRNA, miRNA or ncRNA.
- Cell lines to be transfected (1×106cells, passageable, doubling increasing time is less than 48 hours) and a detailed description of cell culture conditions.
- If there is a need to detect the expression changes of target genes, the appropriate antibody should be offered.
Two approaches for stable cell line screening
- After transfection of plasmid, screen stable cell lines with monoclonal method.
- Screen stable cell lines by lentivirus infection. Compared with plasmid transfection for monoclonal screening, lentivirus infection method is much more convenient and efficient. It is currently the mainstream way to screen stable cell lines.
Transfected stable cell lines can be used to observe functions of target genes and their coded proteins in certain cells.
Application range
- Gene overexpression service
- shRNA interference service
- miRNA overexpression
- Overexpression of any non-coding genes
General protocol:
- Measuring screening concentration: take the antibiotic concentration, which can cause death to all cells within 10 to 14 days, as the screening concentration.
- Cell inoculation: Inoculating cells at the previous day of transfection experiment. Tablets density of various cells are based on their growth rates and cell shapes. Cells density should cover 60% to 80% at the day of transfection.
- Cell transfection (viruses, liposomes, electroporation, FuGENE 6, PEI, etc.)
- Screening cells according to their resistance markers on plasmids.
- Identifying the screening results
Evaluation criteria of screening identification
Usually, qRT-PCR is applied to identify.
Results presented
Construct stable expression cell lines and its related exogenous gene expression analysis system.