Expert Insights | Stable Cell Construction: Lentivirus VS Transposon

Expert Insights | Stable Cell Construction: Lentivirus VS Transposon

In life sciences research, the construction of stable cell lines (stable expression) is a very important technical method. It provides a solid foundation for in-depth exploration of gene function, disease mechanisms, and drug development. Currently, common methods for constructing stable cell lines include lentivirus, transposons, CRISPR-Cas9 gene knock-in, and plasmid transfection. These methods each have their own strengths, providing researchers with diverse choices. Today, follow Ubigene to explore the application scenarios and characteristics of lentivirus and transposon methods.

Construction of Lentivirus-Mediated Stable Cell Lines

Lentivirus, as a powerful gene vector, relies on its strong infectivity and efficient gene integration characteristics, making it the mainstream method for constructing stable cell lines.

1.1Principle

Lentivirus belongs to the retrovirus family, capable of integrating the exogenous gene it carries into the host cell genome, thereby achieving long-term stable gene expression. Lentiviral vectors typically consist of packaging plasmids, envelope plasmids, and transfer plasmids. By co-transfecting these three plasmids into packaging cells, infectious lentiviral particles can be produced.

Figure 1 Schematic diagram of constructing stable cell lines using lentivirus

1.2Construction Process

Vector Construction: Clone or synthesize the target gene and clone it into an appropriate lentiviral vector.

Viral Packaging: Co-transfect the constructed lentiviral vector containing the target gene with packaging plasmids into packaging cells such as 293T. Culture the cells and collect the supernatant containing the lentiviral particles.

Viral Titer Determination: Determine the titer of lentivirus using appropriate methods (such as qPCR or fluorescence assays).

Transduction of Target Cells: Seed the cells to be infected into culture plates and culture them to an appropriate confluency or density. Based on pre-experimental determination of the multiplicity of infection (MOI), add an appropriate amount of lentiviral particles for infection. During infection, polybrene can be added depending on the cell type and virus characteristics to enhance infection efficiency.

Selection of Stable Cells: 48-72 hours post-infection, add the appropriate concentration of selection drugs (such as puromycin, zeocin, etc.) based on the selection marker used. Continue culturing and replace the medium containing the selection drug until all uninfected cells die, leaving surviving cells as preliminarily selected stable cell lines.

Cloning (Optional): Perform further single-cell cloning using the selected cells by limited dilution or flow cytometry to obtain genetically homogeneous stable cell lines.

Validation: Validate the integration and expression of the exogenous gene using methods such as PCR, Western blot, etc.

Storage and Expansion: Freeze and store the selected stable cell lines, and expand them as needed.

Figure 2 Ubigene's Stable Cell Construction Service Workflow

1.3Application Scenarios

Cancer Research: In cancer research, lentivirus is commonly used to construct cell lines stably expressing tumor suppressor genes or oncogenes, studying their effects on biological behaviors such as proliferation, apoptosis, and migration of tumor cells.

Neuroscience: Due to the fact that neurons and other nerve cells are terminally differentiated cells, conventional transfection methods are ineffective, while lentivirus can effectively infect these cells, providing powerful tools for neuroscience research.

Immune Cell Research: For immune cells such as T cells, macrophages, etc., lentivirus can also achieve efficient gene transduction, aiding research on the activation, differentiation, and immune response processes of immune cells.

Construction of Transposon-Mediated Stable Cell Lines

Transposons, as DNA sequences capable of jumping within the genome, provide another unique strategy for constructing stable cell lines.

1.1Working Mechanism

Transposons are a class of DNA fragments that can move within the genome, catalyzed by their encoded transposase, from one location to another. When constructing stable cell lines, the exogenous gene is typically inserted into a transposon vector, and then integrated into the host cell genome through the action of the transposase.

Figure 3 Transposition process

1.2Construction Process

Transposon Plasmid Construction: Insert the target gene into the transposon plasmid, usually adding inverted terminal repeats (ITRs) of the transposon on either side of the target gene.

Co-transfection: Co-transfect the transposon plasmid with a helper plasmid encoding the transposase into the target cells. Transfection methods such as lipofection, electroporation, etc., can be used.

Selection of Stable Cell Lines: After transfection, transposition events occur in the cells, integrating the target gene into the genome. Through appropriate selection methods (such as antibiotic selection), select cells with stable integration of the target gene.

Cloning (Optional): Perform further single-cell cloning using the selected cells by limited dilution or flow cytometry to obtain genetically homogeneous stable cell lines.

Validation: Identify the selected stable cell lines, such as by verifying the integration of the target gene through PCR, Southern blot, and evaluating the expression level of the target gene through Western blot, fluorescence microscopy, etc.

Storage and Expansion: Freeze and store the selected stable cell lines, and expand them as needed.

1.3Application Scenarios

Large Gene Sequence: Due to its advantage of carrying larger payloads, transposons can accommodate larger genes without the need for cDNA truncation. While lentiviral vector has limitations on the size of the inserted gene.

For cells sensitive to lentivirus: If cells are sensitive to viral infection, lentiviral methods may have a negative impact on cell survival, function, and overall condition. In such cases, consider using transposons.

Below is a table summarized by Ubigene for reference:

Summary

Lentivirus and transposons each have their advantages and applications in stable cell line construction, but both suffer from random integration and uncertain copy numbers. CRISPR-Cas9 gene knock-in can serve as an important alternative, but it is costly and time-consuming to produce. Ubigene's EZ-OE™ technology offers a new, more stable, and reliable solution for stable cell line construction. This technology differs from traditional transposon methods by enabling precise genome integration and single-copy insertion, with low cost and a quick turnaround as fast as 5 weeks!

Tandon, N., Thakkar, K. N., LaGory, E. L., Liu, Y. and Giaccia, A. J. (2018). Generation of Stable Expression Mammalian Cell Lines Using Lentivirus. Bio-protocol 8(21): e3073.

Sato M, Inada E, Saitoh I, Watanabe S, Nakamura S. piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs. Pharmaceutics. 2020 Mar 19;12(3):277.

Wei, Mian et al. “Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells.” Frontiers in bioengineering and biotechnology vol. 10 879222. 4 May. 2022, doi:10.3389/fbioe.2022.879222