Introduction to Stable Cell Line Construction Techniques

1. Cell Line Concept

Cell line refers to the population of cells propagated after the first successful passage of the primary cell culture. It also refers to cultured cells that can be passed continuously for a long time. (Hence the later Finite Cell Line and Infinite Cell Line), so a cell line is defined in a narrow sense as a cell that can be passed on continuously (both spoken and written in certain contexts), and in a broad sense as a cell that can be passed on.

Cell lines are cultured cells with special properties or markers obtained from primary cultures or cell lines by selection or clonogenesis. In terms of cultivation algebra, it can be cultivated to 40-50 generations. And special properties or markers of the cell line must be present throughout the culture period. For human tumor cells, cultured in vitro for more than half a year, stable growth, and continuous passage can be called infinite cell lines.

2. Stabilize Cell Lines

The foreign DNA is cloned into a vector with a certain resistance, the vector is transfected into the host cell and integrated into the host chromosome, and the resistance markers contained in the vector are screened to obtain a stable expression of the target protein or a stable expression of the silenced specific gene cell line.

Stable transfection is the integration of foreign genes into the cell's own genome, so that foreign genes become part of the cell genome and can be replicated. Stable cell lines can be screened after stable transfection. Stable cell lines play an important role in recombinant protein/antibody production, gene editing and functional study.

3. Stable Transfection Process

From the perspective of experimental procedure, stable transfection is based on transient transfection: mammalian cells are transfected first, and then the resulting cell pool is screened and finally stable cell lines are obtained. When establishing stable transfection cell lines, we need to use selective markers to distinguish transient transfection from stable transfection, usually with a selective marker in the plasmid, which is co-expressed with the target gene, so that positive clones (foreign genes have been stably integrated into the genome of the cell) can be screened out, while cells that are not stably integrated are eliminated. Finally, stable transfected monoclonal cell lines were obtained by limited dilution.

(1) Cell Recovery

Cell resuscitation is the process of thawing and re-culturing cell lines kept in a liquid nitrogen refrigerator, and resuscitating mammalian cells for subsequent cell transfection. The key to cell recovery is fast melting to prevent ice crystals from forming during the thawing process.

(2) Vector Construction and Cell Transfection

The target gene is constructed into a vector (the vector needs to be resistant), and then the constructed plasmid is linearized. Cell transfection is followed by cell transfection, which can be transfected in a variety of ways, including viral transfection, liposomal transfection, electrotransfer, gene gun method, etc.

(3) Cell Pool Screening

The cell pool can be obtained after transfection. In order to obtain stable transfected monoclonal cell lines, the cell pool needs to be screened: firstly, the positive clones of stable transfection are screened using resistance markers, and then the monoclonal strains are selected by limited dilution method. In addition, if you want to obtain stable cell lines with high expression, it is necessary to perform pressure screening on the cell pool (GS screening system or DHFR screening system), and finally obtain stable cell lines with high expression ability

4. Influencing Factors of Stable Transfection Experiment

(1) Integration probability of exogenous gene: Integration probability of exogenous gene determines the ease of screening of stable transmutation strains;

(2) Copy number: Generally, low copy or single copy can reduce the interference of human factors;

(3) Binding sites: Different integration sites determine the stability of foreign fragments in chromosomes, and some regions are prone to recombination or loss, resulting in loss after screening of stable transmutation strains;

(4) Transcriptional activity of integration sites: The transcriptional activity of integration sites determines the expression quality of foreign gene fragments in stable strains;

5. Comparison of Screening Methods for Different Stable Cell Lines

(1) After transfection of plasmid, monoclonal screening of stable cell lines:

The transfection efficiency was low for most cells. For gene overexpression, if the transfection efficiency reached 40%, the gene overexpression was acceptable. However, for gene interference experiments, the transfection efficiency is much higher than that of gene overexpression, which is usually not satisfied by plasmid transfection.

In addition, the plasmids were free in the cytoplasm and degraded quickly, which could not meet the long-term detection items. Plasmid transfection integration rate is very low, stable cell line construction is time-consuming and labor-intensive, and the yield is very low, often need to select monoclonal strains.

(2) Screening stable cell lines for viral infection:

The virus infection method is more convenient and efficient than the plasmid transfection method for screening monoclonal cell lines, and is currently the mainstream method for screening stable cell lines. Lentivirus is an ideal vector for the Production of stable cell lines due to its high integration, high transcription, high expression, wide host range, high infection efficiency, and integration with cell chromosomes without gene rearrangement.

Lentiviruses can infect almost all kinds of cells, and can integrate into the genome of infected cells after infection for a long period of stable expression, so lentiviruses are often used to prepare monoclonal cell lines that stably express/silence specific genes.

How to Construct stable expression cell lines with lentiviruses:

(1) Suitable antibiotic concentration screening of target cells;

(2) Suitable virus titer screening of target cells;

(3) lentivirus construction and packaging and titer determination;

(4) lentivirus infection;

(5) Screening stable transmissible cell lines for specific antibiotics;

(6) Identification of stable transmissible cell lines.

6. Stably Expressed Cell Lines are Mainly Used in the Following Studies:

(1) Long-term study of gene function in target cells is required. By constructing stable strains, the cost of frequent transfection or virus packaging can be greatly reduced, and long-term experimental studies can be facilitated.

(2) The half-life of some proteins is long, instantaneous RNA can only interfere with expression, and the expressed target proteins cannot be removed. Better gene interference effects can be tested by constructing stable strains;

(3) Unexpected copy number expression (often high instantaneous expression) is introduced by instantaneous transfer, resulting in inaccurate experimental results due to human factors. Building stable strains can help select cells with appropriate copy number for experimental research;

(4) Stable transmutation can be used in the experiment of induced expression system to control the temporal and spatial expression of genes;

(5) Experiments that require the construction of animal models with target cells often require the construction of stable strains.

7. General Service Process of KMD Bioscience Stable Transfer Cell Line Construction Service:

(1) Carrier construction & Virus packaging

In general, the humanized antibody gene is transferred to the lentiviral vector, and then the plasmid expression is detected, and the lentiviral plasmid that overexpresses the target gene is obtained through packaging. The packaging components of the lentivirus vector system and the vector components were transfected into 293 cells for packaging. After 24 to 48 hours, cell supernatants rich in lentiviral particles were collected and concentrated for titer testing.

(2) Cell transfection

The commonly used resistance markers of eukaryotic expression vectors are Puromycin, Hygromycin and Neomycin. First, the screening concentration and viral transfection concentration of Puromycin/G418 were determined, and then the viral suspension was transfected for 24h, and the stable strains of Puromycin/G418 were screened and identified by ELISA and WB.

(3) Monoclonal

The cell pool can be obtained after transfection. In order to obtain stable transfected monoclonal cell lines, the cell pool needs to be screened: firstly, the positive clones of stable transfection are screened using resistance markers, and then the monoclonal strains are selected by limited dilution method.

(4) Cell line stability screening

After several generations of cell lines, genetic instability may occur. We selected the best 10 clones and tested the stability of the cell lines by qPCR and WB after 10 passes. Finally, we provide sequence and vector reports, 3 best-expressing cell lines, expression analysis, stability testing reports, and detailed stable cell line construction reports.

For cells that cannot be passed continuously, our company can also provide cell immortalization services, which can immortalize cells and then transfect and screen them.

This article serves as a reference material for enthusiasts in scientific research. It does not substitute for professional knowledge or hands-on experimental procedures which require more detailed and professional information. In case of any content infringement, 

kindly reach out to the author for immediate deletion of the contentious material.