Lenti-integrated

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Lentivirus-integrated Stable Cell Lines

Definition

Lentivirus：

Lentivirus: Lentivirus is a type of retrovirus belonging to the Retroviridae family. Lentiviruses express reverse transcriptase, which converts viral RNA into double-stranded DNA, and integrase, which inserts viral DNA into host DNA. Lentiviruses divide with host cells. This property allows for the stable delivery of various genes, mutations, or therapeutic drugs into cells, and has been widely used in research for decades. While retroviruses are generally considered to infect only dividing cells, lentiviruses infect both dividing and non-dividing cells.

Wild-type lentiviruses have been engineered into lentiviral vectors that are safe for use in laboratory settings. These engineered lentiviral vectors offer numerous advantages, making them useful research tools in a variety of cell types and models. Lentiviral vectors can target non-dividing cells, enable long-term stable expression in host cells, and exhibit low immunogenicity.

Lentiviral vectors：

The complete lentiviral genome is typically 8-10 kb, encoded by single-stranded RNA, and contains numerous components. However, for safety reasons, many components of the lentivirus used for packaging in experiments have been mutated or removed.

Figure 1. Schematic representation of the complete lentiviral genome.

1.Packaging genes include: gag, pol, env：gag、pol、env
2.Regulatory genes include: tat, rev：tat、rev
3.Accessory genes include: vif, vpr, vpu, nef

Transfer plasmid	LTR	in cis	Long terminal repeat; comprised of a U3-R-U5 structure and are found on each side of the provirus. The U3 (unique 3’) contains sequences necessary for activation of viral genomic RNA transcription. R is the repeat region.
	U3	in cis	Unique 3’; contains sequences necessary for activation of viral genomic RNA transcription. Removal of this region in the 3’ LTR in third-generation plasmids creates self-inactivating viral vectors.
	R	in cis	Repeat region where Tat binds.
	TAR	in cis	Trans-activating response element; found in the R region and acts as the binding site for Tat.
	U5	in cis	Unique 5'; in third-generation plasmids, this region is often removed in 5’ LTRs and replaced with a heterologous promoter (CMV or RSV).
	5' LTR	in cis	Acts as an RNA pol II promoter; the transcript begins, by definition, at the beginning of R, is capped, and proceeds through U5 and the rest of the provirus. Third-generation plasmids use a hybrid 5' LTR with a constitutive promoter such as CMV or RSV.
	3' LTR	in cis	Terminates transcription started by 5' LTR by the addition of a polyA tract just after the R sequence.
	WPRE	in cis	Woodchuck hepatitis virus post‐transcriptional regulatory element; stimulates the expression of transgenes via increased nuclear export.
	RRE	in cis	Rev Response Element; he sequence to which the Rev protein binds.
	Psi (Ѱ)	in cis	RNA packaging signal; recognized by nucleocapsid proteins and essential for efficient viral packaging.
	cPPT	in cis	Central polypurine tract; recognition site for proviral DNA synthesis. Increases transduction efficiency and transgene expression.
Packaging plasmid	gag	in trans	Precursor structural protein of the lentiviral particle containing matrix, capsid, and nucleocapsid components.
	pol	in trans	Precursor protein containing reverse transcriptase and integrase components.
	rev	in trans	Binds to the Rev Response Element (RRE) within unspliced and partially spliced transcripts to facilitate nuclear export. Provided by a separate plasmid from gag/pol in third-generation packaging plasmids.
	tat	in trans	Trans-activator; binds TAR to activate transcription from the LTR promoter. Only in first- and second-generation lentiviral plasmids.
Envelope plasmid	env	in trans	The viral envelope gene; typically vesicular stomatitis virus G glycoprotein (VSV-G), an envelope protein with broad tropism used to pseudotype most lentiviral vectors.

The Evolution of Lentiviral Vectors

For packaging lentiviruses in the laboratory, it is necessary to remove non-essential components and isolate essential components into separate plasmids to ensure safety.

This led to the emergence of four mainstream generations of lentiviral packaging systems:

First-generation plasmids include (Figure 2):
1. Transfer plasmid—contains the transgene and wild-type LTR
2. Packaging plasmid—contains the entire viral genome (packaging, regulatory, and accessory genes), minus only the envelope
3. Envelope plasmid—contains the env.

Figure 2. First-generation lentiviral plasmid system

First-generation lentiviral plasmids have largely been abandoned due to a lack of safety improvements and the inability to produce replication-competent lentivirus (i.e., viral particles capable of infecting cells and further self-replicating).

Second-generation plasmids include (Figure 3):
1. Transfer plasmids—containing the transgene and wild-type LTR
2. Packaging plasmids—containing gag, pol, tat, and rev
3. Envelope plasmids—containing env

Figure 3 Second-generation lentiviral plasmid system

Second-generation lentiviral plasmids significantly improve the safety of lentiviral vector production by removing accessory genes. Second-generation plasmids contain Tat because the 5' LTR of the transfer plasmid serves as a promoter, which requires Tat for activation.

Third-generation plasmids include (Figure 4):
1. Transfer plasmid—contains the transgene and LTR (chimeric 5' LTR)
2. Packaging plasmid 1—contains gag and pol
3. Packaging plasmid 2—contains rev
4. Envelope plasmid—contains env

Figure 4 Third-generation lentiviral plasmid systems

Third-generation systems further improve the safety of second-generation systems in several key aspects. First, the packaging system is split into two plasmids. While safer, this system can be more cumbersome to use and may result in lower viral titers due to the additional plasmids. Second, third-generation systems do not require Tat. Instead, the transfer plasmid contains a chimeric 5' LTR fused to a heterologous promoter (usually CMV or RSV), eliminating the need for Tat transactivation.

Most third-generation transfer plasmids also contain a deletion in the 3' LTR, rendering them self-inactivating (SIN). This deletion is transferred to the 5' LTR after one round of reverse transcription, thereby inhibiting transcription of full-length virus after integration into the host genome. This phenomenon is also common with second-generation transfer plasmids.

Fourth-generation plasmids include (Figure 5):

Figure 5 Fourth-generation lentiviral plasmid system

The fourth-generation packaging system is optimized for viral yield, ease of use, and safety. First, the system utilizes tetracycline (Tet system promoter) for transcriptional activation. Second, it also employs the Tat transactivator to drive high expression of essential viral components. Third, gap-pro and vpr-pol are separated onto two plasmids. This allows for the production of replicative lentiviruses through three low-frequency recombination cycles, which is safer. Titers have been reported to reach 5*10e8 IFU/mL (unconcentrated supernatant).

In summary, after the evolution and upgrades of successive generations of lentiviral vectors, the current fourth-generation packaging system has become the mainstream laboratory lentiviral packaging system, offering high assurance for both safety and yield.

Packaging Host Cells

1. Lentivirus packaging requires the selection of a producer cell line. HEK293T is commonly used. Compared to HEK293, HEK293T cells express the SV40 large T antigen, allowing plasmids containing the SV40 replication origin to replicate episomally, resulting in higher copy numbers. HEK293T cells also have higher transfection efficiency and support higher viral titers.

2. 293T/17 is a subclone of 293T cells that also expresses the SV40 large T antigen. Clone 17 was selected for its higher transfection efficiency in testing.

3. 293FT is also a derivative of 293T cells, a fast-growing variant that also expresses the SV40 large T antigen.

4. Lenti-X 293 cells are also a derivative of 293T cells that express the SV40 large T antigen. It has been reported that Lenti-X 293 cells can package six times more lentivirus than 293FT cells.

5. Suspension 293T cells and various suspension cell derivatives. Suspension cells can achieve higher densities and can be expanded to bioreactors. They are serum-free, facilitating downstream purification and concentration.

6. Stable production cell lines, with the necessary packaging components stably integrated into the host, simplify the production process and ensure batch stability of the virus, eliminating the need for individual packaging required for transient transfection.

Figure 6 Lentivirus packaging diagram

Purification and Concentration

In the next step of lentiviral infection of target cells, high titers and high purity are required, so the collected viral supernatant requires further purification and concentration.
There are various methods for collecting and concentrating viral particles, with common techniques including centrifugation, filtration, precipitation, and immunocapture. The choice of method depends on factors such as the virus type, sample volume, and the desired purity and concentration. Some methods, such as ultracentrifugation, provide high purity but can be time-consuming, while others, such as polyethylene glycol precipitation, are faster but may not achieve the same purity. Newer techniques, such as those using magnetic nanoparticles, allow for rapid, high-yield concentration with minimal handling.

The following is a detailed description of some key methods:
1. Centrifugation:
Differential centrifugation:
This method uses different centrifugation speeds to separate viruses from cellular debris and other components based on size and density.
Ultracentrifugation:
This technique uses extremely high speeds to pellet the virus, thereby concentrating and purifying it.
Centrifugal ultrafiltration:
This method utilizes membranes with specific molecular weight cutoffs to filter out larger particles and concentrate the virus.

2. Filtration:
Tangential flow filtration: This method uses a membrane to separate viral particles from a bulk liquid, enabling continuous flow and concentration.
Membrane adsorption/elution: Viruses can be adsorbed to specific membrane filters and then eluted using an appropriate buffer.

3. Precipitation:
Polyethylene glycol (PEG) precipitation: PEG is used to precipitate viruses from solution for collection and concentration.
Coprecipitation: In some cases, viruses can be coprecipitated with other substances (e.g., proteins) to enhance concentration.

4. Immunocapture:
Antibody-based capture: Viruses can be captured using antibodies that specifically bind to the virus, enabling isolation and concentration.

5. Other methods:
Magnetic nanoparticles: These nanoparticles can be used to capture and concentrate viruses, providing rapid and efficient concentration.
Acoustic separation: This method uses sound waves to separate particles based on size and density, potentially offering a gentle and effective method for viral concentration.
Water-based condensation: This newer technology is under development for sampling viruses in the air.

Titer Measurement

Viral titer is a measure of the concentration of infectious viral particles. Concentrated viruses typically have high titers, so the titer of the concentrated virus must be determined before infecting target cells. Numerous methods exist for measuring viral titer, including plaque assays, foci assays, and various other techniques that quantify viral infectivity or viral particles.

1. Plaque Assay:
This method involves infecting a monolayer of host cells with serial dilutions of a viral sample.
After incubation, visible plaques (areas of cell lysis or death) form, and the number of plaques is counted.
The titer is calculated based on the number of plaques, the dilution factor, and the amount of virus used.

2. Focus Assay:
Similar to the plaque assay, this method quantifies the number of foci (areas of infected cells) formed after infection.
The titer is determined by counting foci and taking into account the dilution and volume.

3. Other Methods:
qPCR: Quantifies viral RNA or DNA, providing a measure of total viral particles.
ELISA: Detects viral antigens, providing a method for quantifying viral load.
Flow cytometry: Can be used to count single infected cells, especially those expressing fluorescent markers.

Figure 7. Products present in lentiviral packaging supernatant

Physical methods can measure functional and nonfunctional particles released into the supernatant, as well as free capsid proteins (e.g., p24). Functional titration methods specifically measure functional viral particles.

Important points to note:
1. Infectious titer vs. physical titer:
Some methods measure the number of infectious viral particles (infectious titer), while others measure the total number of viral particles (physical titer). Infectious titer better reflects the virus's ability to infect and transduce. However, detecting infectious titer generally takes longer, while detecting physical titer is faster. Therefore, one type of method establishes a conversion relationship between "infectious titer" and "physical titer."

2. What is the difference between plaque-forming units (PFU), transduction units (TU), and infectious units (IFU)? Which one better reflects the amount of active virus used?
1) PFU (plaque-forming unit) indicates the number of infectious or live virus. PFU/ml is used to calibrate viral titer by measuring the number of plaques formed after virus infection and reflects the amount of active virus in the preparation.
2) IFU (infectious unit) is equivalent to PFU.
3) TU/ml: transduction units/ml, indicates the number of biologically active viral particles per milliliter of viral venom, i.e., the number of viral genomes capable of infecting and entering target cells. This is a commonly used unit of measurement for lentiviruses and reflects the amount of working virus in a preparation.
The number of IFU is typically lower than the number of TU because not all viral particles that enter cells successfully initiate productive infection.

Infecting Target Cells

Lentiviral transduction involves integrating the target gene into the host genome. This is achieved by modifying the reverse transcriptase and integrase enzymes encoded within the lentiviral vector, enabling sustained expression of the exogenous gene. Unlike other viral vectors, lentiviruses can transduce non-dividing cells, which offers unique advantages in gene delivery applications.

In establishing stable cell lines, lentiviruses infect host cells. The reverse-transcribed DNA remains partially free in the cells, while the integrase enzyme integrates the 5' to 3' LTR sequences of the lentiviral vector into the host genome. Under selective resistance screening, non-integrated cells are killed, and surviving cells can be expanded to establish stable cell lines. Studies have shown that it takes approximately 10-14 days for the non-integrated DNA to be completely degraded as cells divide, which is also the period of pressure screening for resistance.

Lentiviruses (LVs) are enveloped viruses, the most common of which is VSV-G. They enter cells by binding to specific cell surface proteins (such as LDLR), then undergoing membrane fusion and injecting viral RNA containing their genetic information into the cytoplasm. This single-stranded viral RNA is converted into DNA by reverse transcription. The viral DNA enters the cell nucleus and is integrated into the host cell genome by the viral integrase.

Figure 8 Schematic diagram of lentivirus integration into the host genome

Key Considerations in Experiments

1. MOI Selection: An MOI that is too low will result in low transduction efficiency, while an MOI that is too high may cause cytotoxicity. MOI titration experiments are necessary to determine optimal infection conditions.

2. Polybrene Use: Polybrene is a polycation that reduces charge repulsion between the virus and the cell membrane, thereby improving transduction efficiency. However, if the dosage is inappropriate, it may also cause cell damage.

3. Timing: Transduction should be performed as soon as cell viability has recovered, ensuring that cells are in an active proliferation phase as soon as possible.

4. Cell Density Control: Excessively high cell density increases transduction costs, while excessively low density may affect efficiency. It is recommended to maintain a cell density of approximately 1-2 × (10^5) cells/mL.

5. Viral Concentration and Titer: Lentivirus titer directly affects transduction efficiency, emphasizing the importance of understanding viral titer and controlling viral addition.

6. Integration: Genome-wide studies of viral integration have shown that lentiviruses most frequently integrate into transcriptionally active regions, regions recently involved in translocation events, and other "vulnerable" genomic locations, and this preference is conserved across target species. While the general tendency toward integration is known, it remains random and difficult to predict.

Finally:
Due to their ability to integrate and maintain long-term transgene expression, lentiviral vectors are valuable research and clinical tools. Common applications include stable cell line development, CRISPR library delivery, mouse modeling, and gene therapy.
Lentiviral transduction is a stable and reliable gene delivery method suitable for a variety of cell types, including non-dividing cells that are often resistant to other transduction methods. Careful experimental design and optimization can significantly improve transduction efficiency and subsequent stability of gene expression.

Stable Cell Line Construction Services

CB-Gene offers stable cell line construction services using the "Lentiviral Integration System." Using this system, we have successfully constructed over 900 stable cell line models and have extensive experience. We welcome inquiries.

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