Common Problems and Solutions in Stable Cell Line Construction

Stable cell lines are ones that have the ability to either interfere with a certain gene's expression or express a particular gene persistently. Establishing stable cell lines can help compensate for the short expression duration of foreign genes in transient transfection and allow long-term monitoring of protein interactions and the effects of genes on cell function in the research of gene function.

The integration of foreign DNA into the cell's genome incorporates it into the cell's genetic material, enabling replication. Establishing stable cell lines is essential for various applications such as recombinant protein production, antibody generation, gene editing experiments, and functional investigations.

KMD Bioscience can produce a range of lentiviral vectors that target certain genes based on the needs of the client. These vectors include gene site knockout (KO), gene knockdown (KD), and gene overexpression (OE and Cas9-SAM). Moreover, we can produce lentiviral particles that meet specific purity standards (≥1E+8 IU/ml). These particles can infect target cells, which can subsequently be screened to produce stable cell lines tailored to meet the needs of customers for scientific or medical research.

Q1: How to select expression vectors when constructing cell lines?

A: During the process of incorporating target protein genes into expression vectors, it is common practice to choose vectors that contain promoters, terminators, and selective markers, such as antibiotic tolerance genes.

The appropriate vector is selected based on vector resistance, promoter type, solubility, and expression of fluorescent proteins (e.g., GFP/RFP), and the need to screen for stable transformation in plants.

Q2: How to improve construction of stable cell line transfection efficiency?

A: Using transgenic technology to obtain objective characteristics of transgenic plants, is a powerful way to the improvement of crop varieties, but the phenomenon such as gene silencing have reduced the expression efficiency of foreign genes in transgenic plants.

Transfection was performed using high-quality plasmids, including lentiviral packaging plasmids and lentiviral vectors. Once transfection is complete, the sample is placed in an incubator. For biosafety reasons, the incubator used for lentivirus packaging should be separated from the normal cell incubator and maintained under the same culture conditions.

Q3: What are the differences between monoclonal and polyclonal cell lines in the construction of stable cell lines?

A: Monoclonal antibody technology is an important tool in modern life science research, it plays an indispensable role in the structure and function of genes and proteins, and still plays an irreplaceable important role in the immunological diagnosis of humans, animals and plants.

Techniques for the preparation of monoclonal antibodies continue to evolve, but each technique has its advantages and disadvantages, so resources need to be integrated. Antibody library technology can utilize wild-type or genetically modified animals that have been immunized, or it can use single B cells that have been screened from patients who have recovered from a particular disease. In short, these preparation techniques can be applied cross-over.

The polyclonal cells were obtained directly by transfecting the target gene followed by screening for resistance genes. They made up by multiple positive cell clones.

Q4: How to determine drug concentration during stable cell line screening?

A: Although the addition of drugs for stable cell line screening helps to screen for highly productive, stable cell lines, excessively high drug concentrations often inhibit cell growth and adversely affect the cells. The appropriate screening pressure concentration needs to be determined based on the experimental results. Many companies now offer platform cell lines that can be used directly with their recommended screening pressures.

Q5: Stable cell lines can be used in various ways?

A: Monoclonal antibodies are used in a wide range of fields, including medicine, agriculture, food and the environment. In basic research, protein purification, environmental and food monitoring, disease diagnosis, prevention and treatment and eugenics, monoclonal antibodies have irreplaceable important roles.

It can be used in the construction of immortalized cell lines, gene knockout stabilized cell lines, and as a good basic preparation for subsequent scientific research.

Construction of immortalized cell lines is an ideal cell model to study the mechanism of cell proliferation, differentiation, apoptosis and cell senescence. Immortalized cell lines are cell lines that can proliferate without restriction in vitro without aging when subjected to external stimuli such as physical or chemical stimulation, genetic mutation, or viral infection.

Gene knockout stabilized cells refer to the application of CRISPR technology for gene editing. This process involves designing a specific single guide RNA (sgRNA) to target the cutting site of the gene of interest. Subsequently, the Cas9 protein binds to the sgRNA and removes the target gene from the cell, resulting in gene knockout.

Gene knockout cell line is an important means to study gene function and achieve precise deletion of gene. The most recent development of prokaryotic knockout cell lines is the gene knockout technique in bacteria using the Red system in phages, but it is mostly carried out in E. coli. Eukaryotic knockout cell lines are the fastest developing PCR-mediated yeast gene interruption technology and RNAi interference technology, which greatly simplify the experimental operation and shorten the experimental time, and have attractive application prospects in the field of studying the function of unknown genes and proteins in biological genomes.

KMD Bioscience has established a comprehensive cell biology technology platform and a standardized cell laboratory, along with a diverse and extensive cell bank resource. We can provide you with a variety of cell lines to develop services, including silent stable cell line, stable expression cell lines, etc. All these cells are certified by STR analysis.

Our technical team members strictly control every step of the experiment to effectively increase the production capacity of stable cell lines, improve experimental results, and ensure product quality. The stable cell lines we prepare can be utilized in various research fields, such as recombinant protein and antibody production, gene editing, and functional research.

References

[1] Hsiao MH, Miao Y, Liu Z, et al (2022). Molecular Display of the Animal Meta-Venome for Discovery of Novel Therapeutic Peptides. Preprint. bioRxiv. 56(9), 4569-4582.

[2] Benard-Valle M, Wouters Y, Ljungars A, et al (2024). In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model. Nat Commun.15(1):4310.

[3] Chen, P. C., Guo, X. S., Zhang, H. E., et al (2024). Leveraging a Phage-Encoded Noncanonical Amino Acid: A Novel Pathway to Potent and Selective Epigenetic Reader Protein Inhibitors. ACS central science, 10(4), 782–792.