Cell Line Construction Overview

Stable cell lines refer to those that possess the ability to sustainably exhibit a designated gene or disrupt the manifestation of a specific gene. In the study of gene function, the establishment of stable cell lines can compensate for the limited expression time of foreign genes in transient transfection and enable long-term observation of protein interactions and the impact of genes on cell function.

KMD Bioscience offers a range of cell line construction services and lentiviral vectors tailored to target specific genes according to customer requirements. These vectors include gene overexpression (OE and Cas9-SAM), gene knockout (KD), gene site-knockout (KI), and gene site-knockout (KO). Additionally, we can produce lentiviral particles meeting different purity standards (≥1E+8 IU/ml). These particles can be used to infect target cells, and subsequent screening can be done to establish stable cell lines tailored to meet the requirements of customers for scientific research or clinical experiments (These included silent stable cell lines, overexpression stable cell lines, and so on).

Construction of Stable Cell Lines: Principles

Integrating 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.

Stable Cell Line Screening in the Construction of Stable Cell Lines

The stable cell lines can be categorized into primary cells and passaged cells.

Primary cell culture refers to the process of culturing tissues or cells in vitro for the first time. The culture is continuously maintained in an in vitro growth environment without undergoing segmentation. Initially, various exogenous immortalization genes are introduced into target cells through cell immortalization methods, disrupting the normal cell division cycle and granting them unlimited proliferation capacity. Reaping precise information can be facilitated by nurturing primary tumor cells obtained from patients. This process assists in translating in vitro findings into in vivo models and, ultimately, clinical implementations. A passaged cell culture is acquired by cultivating primary cells, resulting in the propagation of a homogeneous population of cells that can be replicated multiple times consecutively. Over a finite number of passages, these cells undergo changes in their characteristics, experience mutations, and eventually cease to exist. Diploid cell lines exhibit a limited lifespan.

Passage culture refers to dividing the culture into smaller parts and then reintroducing it into other cultural contexts for further development. Passaged cell lines are an integral part of current research on malignant tumors. These cell lines can be used in monolayer cultures or as part of three-dimensional (3D) tumor models. When transplanted into immunocompromised animals, they can also be used to model the tumor microenvironment in vitro or in vivo. 

Methods for Constructing of Stable Cell Lines

I. Lentiviral Infection System: The gene delivery system, developed based on HIV, can infect both non-dividing and dividing cells. It efficiently integrates foreign genes into the host chromosome, enabling long-lasting expression of foreign genes with high safety.

II. Transposon System: In this system, two transposon elements are separated and placed on their respective vectors. The original transposon enzyme sequence is replaced with the gene sequence of interest. The other vector independently expresses the transposition enzyme, facilitating the integration of the target gene into the host genome when co-transfecting the target cell.

III. CRISPR knock-in system: There are two ways to achieve this. The first method involves site-specific knock-in, which necessitates designing specific sgRNA sequences and donor sequences for introducing site-specific mutations. The second approach is to integrate foreign genes into predetermined sites within the target cell.

IV. Plasmid screening for stable transfer cell lines involves the successful introduction of only the target plasmid into the cell. Subsequently, the corresponding resistance is utilized for long-term screening to achieve a stable transformation of the cell line.

Table 1. Comparison of Cell Line Construction Methods

The General Process of Constructing Stable Cell Lines

I. Screening appropriate antibiotic concentrations and viral titers for use in target cells.

II. Lentivirus infects target cells.

III. Pressure screening.

IV. Stable Cell Line Screening.

V. Detection of stable transmissible cell lines.

Figure 1. Cell Line Construction

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 lines, 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.