It is well known that cell lines play an important role in gene function studies, drug screening, and the production of recombinant proteins and antibodies. Herein, we have collected the Frequently Asked Questions on cell line generation, hoping to help researchers gain a proper understanding of how cell lines are developed and used.


What is the difference between transient and stable transfection?

In stable transfection, the plasmid DNA integrates into the cellular genome and will be stably passed on to future generations of the cell. In transient transfection, the foreign genes do not integrate into the cell genome, so cells express the transiently transfected gene for a short period of time. Notably, most foreign genes will be lost with cell division in transient transfection. Researchers are unable to achieve the purpose of continuous expression of foreign genes with transient transfection.


Which transfection method should we choose? Stable transfection or transient transfection.

Reasons to choose stable transfection:

  • The experiment requires a long-term gene function study in target cells.
  • The half-life of some proteins is very long, transient RNA can only interfere with the expression but cannot remove the expressed target protein, so we need to achieve a better gene interference effect.
  • To make the experiment more accurate, cells with appropriate copy numbers for experimental research may be used.
  • The inducible expression system can be used to overcome embryonic lethal gene modifications and provide control over temporal and spatial expression.
  • Cells will be used for animal experiments, such as tumor formation in nude mice.


Reasons to choose transient transfection:

1. Study the target gene overexpression or interfering effects in a short time.

2. Transient expression for 2-4 days may already achieve specific experimental purposes, such as testing for interactions between two exogenously transfected proteins of interest.

3. Individual cell differences have an impact on experimental results.


What is gene overexpression, interference, or gene knockout? 

Gene overexpression refers to the process of forcing the expression of the gene to a much higher than normal level, i.e., the gene is over transcribed and/or translated so that the final gene expression product exceeds the normal level. Since overexpression requires the expression of a long chain of mRNA, it is more difficult to achieve than interference, which only needs to express a short shRNA fragment.

Gene interference (a.k.a. gene knockdown) is used to interfere with the activity of a specific gene by altering the gene regulatory or coding sequences either with genetic modification or by treatment with a reagent, such as an RNA oligonucleotide.

Gene knockout results in the permanent inactivation of the target gene in all cells of the body throughout development.


How should researchers choose between these model types?

Using gene overexpression, we can obtain a great number of target gene products. These target gene products can be used for research or production, such as studying the 3D structure of proteins or preparing insulin using bioproduction technology.

Interfering with a stable transgenic strain can meet the experimental needs regarding down-regulation of gene expression. However, interfering with stable strains does not control where the shRNA is inserted into the chromosome, shRNAs on the chromosome are sometimes lost. In addition, shRNAs have off-target effects that may interfere with other unknown genes. The shRNA is acting with the mRNA of the gene of interest to degrade it, which is decreasing the expression of the gene of interest at the RNA level but does not eliminate protein expression.

Stable knockout strains are engineered at the genomic level by loss of gene function through changes in the gene sequence, so the expression of the target protein is usually not detected in knockout cells. Since the knockout is stable and cannot be restored, which means the gene is a permanent knockout and the model will have minimal problems with residual expression. However, developing a stable gene knockout strain comes with the disadvantages of a long experimental cycle, expensive, and low efficiency. We should have comprehensive consideration when choosing a down-regulation method in cell line experiments.


How do you choose colonies for stable cell lines?

When it is necessary to remove the potential interference factors of the cell population, it is recommended to use a monoclonal stable cell line, which has a relatively pure genomic background and low interference factors to the experimental results. When we study system biology, we need to consider the factors of the cell population. At the same time, it is also due to the large individual differences between different cells, and the monoclonal cell lines with different integration sites may show inconsistent cell behaviors, so many experiments use mixed clones to better eliminate interference caused by differences between cells. Therefore, the choice of monoclonal cell line or mixed clone cell line depends on the purpose of the experiment.


How to identify and screen monoclonal cell lines from stable cells?

If the foreign fragment contains a fluorescent label, you can directly use the flow cytometer to detect whether the fluorescence is uniform. Additionally, immunofluorescence labeling can be used alongside flow cytometry to observe whether the fluorescence distribution has a uniform peak.

If the foreign fragment does not contain any label or fluorescent label, it can be identified by molecular biology methods such as Southern blot. In addition, 96 well plate dilution method can be used to screen the positive clones, and the positive clones obtained are generally monoclonal.


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