Stable Cell Line Construction Protocol and FAQs

A stable cell line can stably express the target gene for a long time without selection pressure by integrating the exogenous gene into the genome of the host cell. Compared with transient transfection, stable cell lines can not only provide consistent and repeatable experimental results, but also greatly reduce the variability of experiments. It is an important tool for biological research and industrial application.

At the beginning of the 20th century, scientists had begun to try to culture cells in vitro, which laid the foundation for later cell line construction. In the middle of the 20 th century, cell culture technology gradually matured, and Henrietta Lacks ' HeLa cells became the first widely used human cell line, marking the beginning of immortalized cell line research. Scientists began to explore the method of introducing exogenous DNA into cells. Most of the experiments in this period were transient transfection without stable integration. In the late 1997 s, with the development of molecular cloning technology, scientists were able to better manipulate DNA sequences. This period also witnessed the application of antibiotic selection marker system in the construction of stable cell lines. Tools such as SV40 virus are used to increase the expression of exogenous DNA in mammalian cells. In 1998, people began to use plasmid vectors and viral vectors for genome integration. Technological advances in this period have enabled target genes to be more effectively embedded in the host genome. The introduction of resistance genes (such as neomycin resistance gene neo) makes it possible to select cells carrying foreign genes through drug screening.In 1999, the success rate of exogenous gene transfection was greatly improved by the wide application of efficient DNA introduction methods such as liposome-mediated transfection and electroporation. At the same time, the development of flow cytometry has also promoted the screening of monoclonal cells. Since the 21 st century, with the rise of gene editing technologies such as CRISPR-Cas9, the accuracy of stable cell line construction has been greatly improved, and scientists have been able to perform gene insertion and knockout more specifically. In addition, the development of enhancer recognition and synthesis, optimized promoters, etc., has improved the expression level and stability of foreign genes.

The Steps of Stable Cell Line Construction

Vector Construction

First, select the appropriate target gene according to the research objectives. Gene sequences were analyzed by bioinformatics tools to ensure that they were suitable for expression without strong secondary structures or sequences that were not conducive to transcription and translation. In order to facilitate the subsequent cloning operation, specific restriction sites were designed at both ends of the target gene, which could match the multiple cloning sites of the vector in the subsequent construction.

The vector and the target gene fragment were digested with restriction endonuclease, and the target gene was inserted into the multiple cloning site of the expression vector by T4 DNA ligase. The ligated product was transformed into E.coli to obtain multiple recombinant colonies. After PCR screening of colonies or small amount of plasmid extraction, positive clones were selected for sequencing verification to ensure that the target gene was correctly inserted and there was no mutation.

Cell Transfection

The host cell line suitable for the experiment needs to be selected. Usually, the cells need to be transfected in the logarithmic growth phase to ensure that they have the best state and efficient DNA uptake ability. The cell density should reach a confluence of 50-70 %, which is the recommended density for most transfection reagents and can be adjusted by cell counter or microscope observation. Then, the appropriate transfection method was selected according to the cell type. For adherent cells, liposome-mediated method is commonly used ; suspension cells may require electroporation. To optimize the transfection conditions, variables such as DNA concentration, cell density, and medium type need to be considered. The optimal conditions were determined by setting up a control experiment.

Resistance Screening

In general, 24-48 hours after transfection, the cells have recovered well, and an appropriate amount of selective antibiotics were added for screening. The selection of antibiotics is based on the resistance genes contained in the vectors used, such as neomycin (G418), puromycin, etc. In order to ensure the successful integration of resistance genes, it is necessary to continuously observe and regularly replace the medium containing antibiotics. This process usually lasts for 1-2 weeks until all the untransfected control cells die, and the transfected cells survive and grow normally. During the period, dry medium should be avoided and antibiotics should be supplemented in time.

Cloning Selection and Amplification

Once the resistant population cells are obtained, the next step is to perform monoclonal sorting on these cells. The limited dilution method is to dilute the cells to extremely low concentrations and distribute them in a 96-well plate to ensure that each well contains at most one cell. Or flow cytometry can be used to sort single cells more accurately. The selected monoclonals need to be amplified under standard culture conditions. Each step in this process needs to avoid cross-contamination, carefully label each clone and record its growth status. After several rounds of amplification, a stable cell line can be obtained.

Gene Verification

The integration of the target gene was detected by PCR by extracting the genomic DNA of the candidate clones. PCR amplification can preliminarily confirm whether the target gene exists and integrates into the host genome. After confirming gene integration, its expression level needs to be further verified. qPCR was used to detect the expression of target gene mRNA and provide quantitative results. Western blot is used to detect protein levels to ensure that post-transcriptional genes can still be effectively translated into proteins. After ensuring that these indicators meet the experimental requirements, the cell line can be identified as a candidate clone for stable expression.

Figure 1. Strategy for generation of pseudotyped packaging cell line. (Reference source: A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.）

application of Stable Cell Line Construction

Basic Research

For gene function research, signal pathway analysis and epigenetic research.

Biopharmaceuticals

Large-scale production of therapeutic proteins, such as monoclonal antibodies, hormones, etc.

Drug Development

Drug target verification, efficacy evaluation.

In today 's life science research, stable cell lines as a very important tool, is widely used in drug screening, gene function analysis and protein expression and other fields. Alpha Lifetech provides stable cell line construction services and tailor-made solutions for global customers.

FAQ

1. Low transfection efficiency.

Problem description : Low transfection efficiency will lead to insufficient number of positive clones, which will affect subsequent screening. Solutions : (1) Optimize the transfection conditions : adjust the DNA concentration, cell density and the amount of transfection reagent. (2) Select the appropriate transfection method : for example, use electroporation to improve the efficiency of some difficult-to-transfect cell lines. (3) Use efficient transfection reagents : For specific cell types, try different commercial transfection reagents.
2. Low survival rate of resistant clones.

Problem description : In the antibiotic screening phase, some or all cells died. Solutions : (1) Optimize the concentration of antibiotics : First, gradient experiments were performed to determine the minimum effective concentration to avoid non-specific cytotoxicity caused by too high concentration. (2) Prolong the recovery time : Let the cells recover for a period of time without selective pressure before starting antibiotic screening.
3. Gene expression level is unstable or too low.

Problem description : Even if the positive clones were successfully screened, the expression level of the target gene did not meet the experimental requirements. Solution : (1) Select the appropriate promoter : select a strong promoter according to the characteristics of the host cell to improve the expression level. (2) Multi-copy integration : Increase the copy number of the integrated gene by increasing the vector concentration or using a vector with a multi-copy insertion site. (3) Select the appropriate cell lines : Some cells themselves have natural inhibition of the expression of foreign genes, you can try to replace the cell line.
4. Cloning purity problem.

Problem description : The obtained monoclonal cells may be heterogeneous and contain a variety of genetic backgrounds. Solution : (1) Repeated dilution separation : Limited dilution or flow cytometry (FACS) was used for multiple sorting to ensure that each clone was derived from a single cell. (2) Genotyping : Each clone was verified by PCR or sequencing to confirm that it was a single clone.
5. Poor cloning stability.

Problem description : With the increase of the number of passages, the expression level of the target gene gradually decreased. Solutions : (1) Low passage culture : minimize the number of passages and complete subsequent experiments at lower passages. (2) Periodic screening : regularly cultured under selective conditions to maintain the selection pressure of resistance genes. (3) Use more stable expression systems : for example, select systems embedded in homologous recombination fragments to achieve more stable gene integration.

reference

[1] Chen LQ, Wang YN, Li DY, et al. Construction of a Stable Expression Cell Line of Human Phospholamban. Fa Yi Xue Za Zhi. 2021;37(5):615-620. doi:10.12116/j.issn.1004-5619.2020.400909

[2] Lü X, Zhou Z, Zhu L, Zhou J, Huang H, Zhang C, Liu X. [Construction and identification of a HEK293 cell line with stable TrxR1 overexpression]. Nan Fang Yi Ke Da Xue Xue Bao. 2022 Apr 20;42(4):554-560. Chinese. doi: 10.12122/j.issn.1673-4254.2022.04.11.

[3] Ory DS, Neugeboren BA, Mulligan RC. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci U S A. 1996;93(21):11400-11406. doi:10.1073/pnas.93.21.11400

[4] Wei LY, Lu N, Meng L, Li XJ, Li L, Li CK, Li DL. [Construction of eukaryotic expressing vector of human P2X7 and establishment of stable transfectant cell line]. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2016 May 8;32(5):471-475. Chinese. doi: 10.13459/j.cnki.cjap.