Advances in Overexpressing Cell Line Construction Technology and Analysis of Quality Assessment Systems

Comparison of Mainstream Construction Technologies and Their Characteristics

The construction technologies for overexpression cell lines have undergone years of development and have formed multiple types of mature tools. Lentiviral vector systems have become one of the commonly used methods in laboratories due to their high infection efficiency and ability to integrate into the host genome for long-term stable expression. These vectors can carry relatively large exogenous gene fragments and are particularly suitable for hard-to-transfect cells (such as primary cells and suspension cells). Another class of transposon-based systems (such as PiggyBac and Sleeping Beauty) has also attracted attention, with advantages including stable maintenance of the carried genes after multiple passages and relatively random insertion sites, which helps avoid genomic silencing effects. For research scenarios suitable for transient high expression, plasmid transfection combined with antibiotic selection can also rapidly yield stable pools, but it should be noted that cell lines obtained by this method have poor expression uniformity and usually require monoclonal purification. In addition, CRISPRa (CRISPR activation) technology can endogenously upregulate the expression of specific genes and avoid ectopic regulation of exogenous genes; however, due to its complex regulatory mechanism, classical cDNA overexpression remains the primary approach in routine overexpression cell line construction.

Standardized Procedures for Selection and Monoclonalization

After obtaining a stably transfected population, selection is a key step to ensure expression uniformity. Generally, drug resistance genes co-expressed with the vector (such as puromycin resistance gene and blasticidin resistance gene) are used for pressure selection, usually lasting 7-14 days, during which fresh medium containing antibiotics needs to be replaced multiple times. After completing population selection, monoclonalization by limiting dilution or flow sorting is recommended. Limiting dilution is simple to perform, but requires seeding approximately 0.5 cells per well in a 96-well plate and picking monoclonal colonies after 10-14 days of culture. Flow sorting can directly sort single cells into 96-well plates based on fluorescent reporter genes or surface markers, offering higher efficiency but also higher cost. Regardless of the method used, each clone subsequently needs to be verified for expression level. Generally, qPCR is used to detect relative mRNA expression, and Western blot or flow cytometry is used to confirm protein expression. Only clones with stable expression and no cytotoxic characteristics are suitable for long-term cryopreservation and use.

Evaluation of Expression Stability and Batch Consistency

Overexpression cell lines may exhibit gene silencing or decreased expression during long-term passaging, which is mainly related to the chromatin state at the exogenous gene integration site, promoter methylation, and other factors. Therefore, establishing a comprehensive stability monitoring system is the core of quality assessment. It is recommended to test expression levels at the following time points: after selection is completed (T0), after 10 consecutive passages (T10), and after 20 passages (T20). If the expression level at T20 decreases by more than 30% compared with T0, re-selection is required or consideration should be given to replacing the commonly used CMV promoter with other promoters (such as EF1α and CAG), since the former is more prone to silencing in certain cell lines. At the same time, each batch of working cell banks produced should be tested for mycoplasma, bacterial, and fungal contamination before use, and it is recommended to preserve a sufficient number of early-passage seed banks. For overexpression cell lines used in in vivo tumor models, it is also necessary to evaluate the maintenance of expression in tumor tissues after xenotransplantation, because the in vivo environment may accelerate gene loss.

Common Problems and Targeted Solutions

Problems such as cytotoxicity, polyclonal silencing, and target protein misfolding are often encountered during the construction of overexpression cell lines. If cytotoxicity occurs, it may result from the target protein having a naturally harmful effect on host cells or from excessively high expression. In this case, attempts can be made to switch to a weaker promoter, reduce the selection concentration, or use an inducible expression system (such as Tet-on). If the positive rate of cell population expression after selection is low, it is recommended to optimize transfection/infection parameters, including increasing viral titer or changing the delivery method. Misfolding of the target protein may affect functional experiments; consideration can be given to adding a signal peptide, co-expressing molecular chaperones, or using a C-terminal tag (such as HA or Flag) to assist detection, but care should be taken to avoid tag interference with protein activity. In addition, some cell lines (such as Jurkat and THP-1) are difficult to transfect, and polybrene or spinoculation should be used during lentiviral infection to improve efficiency.

Conclusion

The construction of overexpression cell lines has gradually moved from small-scale laboratory operations toward standardized workflows. Reasonably selecting the technical route and strictly implementing quality assessment are the basis for ensuring the credibility of downstream experiments. Whether used for basic mechanistic research or industrial-grade screening, stable overexpression cell lines are indispensable cell model tools.