Lentivirus is an enveloped RNA virus with a diameter of 80–120 nm, exhibiting an icosahedral symmetric structure and a spherical shape. The outermost layer of the viral particle is the envelope (the envelope protein determines the type of cells the virus can infect), followed by the matrix protein and capsid from the outside in. The innermost part contains two identical positive-sense RNA strands and enzymes (reverse transcriptase, integrase, and protease).Lentiviral expression vectors contain the genetic information required for packaging, transfection, and stable integration. Lentiviral packaging plasmids can provide all the helper proteins needed for packaging recombinant lentiviral vectors. To generate high-titer viral particles, it is necessary to co-transfect cells with both the expression vector and packaging plasmids simultaneously. Viral packaging occurs within the cells, and the packaged pseudoviral particles are secreted into the extracellular culture medium. After centrifuging to obtain the supernatant, it can be used directly or after concentration to infect host cells.The first step of lentiviral infection of cells is the binding of the envelope protein to receptors on the cell surface. After the lentivirus fuses with the host cell membrane, it releases structural proteins, enzymatic proteins, and the viral core. Under the action of reverse transcriptase, the viral RNA undergoes reverse transcription and forms a pre-integration complex with integrase. After the pre-integration complex enters the cell nucleus, integrase catalyzes its integration into the host genome. The integrated DNA is transcribed into mRNA, which is transported back to the cytoplasm to express the target protein or induce RNAi interference (see figure below).
The Lentiviral Packaging Kit (10T) belongs to the second-generation lentiviral packaging system and is compatible with both second-generation and third-generation lentiviral packaging plasmids. The toxic genes in the lentivirus have been eliminated and replaced with exogenous target genes, making it a pseudotyped virus. Specifically, after the generated lentivirus infects target cells, it will not infect other cells, nor will it use host cells to produce new viral particles. The lentivirus packaged by the Lentiviral Packaging Kit (10T) features high infection efficiency and a broad infection spectrum, enabling stable and sustained expression of exogenous genes in host cells.
The Lentiviral Packaging Kit (10T) contains the following components:
1. Optimized Proportion Lentiviral Packaging Helper Plasmid Mix (Package Plasmid Mix)Designed and adjusted to be compatible with most lentiviral expression vectors, ensuring that recombinants are packaged into lentiviral particles with high infectivity.
2. Control Plasmid Expressing GFP and Puro Markers (Control Plasmid)Facilitates observation of viral packaging efficiency and determination of viral titer.
3. High-Efficiency Transfection Reagent (Lentivirus Transfection Reagent)Its optimized formula is particularly suitable for viral packaging in lentiviral packaging cell lines (e.g., HEK293T, HEK293FT). It features high efficiency, low toxicity, simple operation, and good repeatability—these advantages can significantly improve the packaging efficiency of lentiviral vectors in lentiviral packaging cell lines and produce recombinant lentiviruses with higher titers.
4. Common Viral Infection-Enhancing Reagent (Polybrene) Can significantly improve the contact between virus and cells as well as infection efficiency.
Product Components and Storage Conditions:
L1455942
| Component
| 10T | 20T | 40T | Storage
|
L1455942A
| Package Plasmid Mix
| 40 μL | 80 μL
| 160 μL
| -20℃ |
L1455942B
| Control Plasmid (1μg/μL)
| 10 μL
| 20 μL
| 40 μL
| -20℃
|
L1455942C
| Lentivirus Transfection Reagent
| 400 μL
| 800 μL
| 1600 μL
| -20℃
|
L1455942D
| Polybrene (10mg/mL)
| 200 μL
| 400 μL
| 800 μL
| -20℃ |
Product Features:
⁕ Simple and Fast – The operation is simple, with no need for extraction of packaging helper plasmids or optimization of the packaging system.
⁕ High Viral Titer – Capable of packaging lentiviruses with high titer and high expression levels.
⁕ Wide Application Range – Designed and optimized for proportion, it is compatible with most lentiviral expression vectors.
Summary of the Experimental Procedure:
Figure 1. Experimental Flowchart of the Lentiviral Packaging Kit (10T)
Experimental Steps:
I. Preparation of Recombinant Lentivirus
1. Cell Preparation
One day before transfection, seed 4–6×10⁶ lentiviral packaging cells (e.g., HEK293T, HEK293FT) into a 10 cm cell culture dish. Incubate in a 37°C, 5% CO₂ incubator for 16–24 hours, ensuring the cell confluency reaches 70%–80% at the time of packaging.
⁕ Cell status significantly affects viral packaging efficiency. Packaging cells should be in good growth condition; it is recommended to use cells in the exponential growth phase with a viability >90% for lentiviral packaging.
⁕ When used in conjunction with the Lentivirus Packaging Cell Line, this packaging system can produce lentiviruses with higher titer and higher expression levels.
2. Lentiviral Packaging
(1) Add 4 μL of the Lentiviral Packaging Helper Plasmid Mix (Package Plasmid Mix) to a 2 mL centrifuge tube. Add 4 μg of the lentiviral vector plasmid containing the target gene, mix gently, then add 40 μL of Lentivirus Transfection Reagent to the plasmid mixture. Incubate at room temperature for 3 minutes.
⁕ The lentiviral vector plasmid must be constructed according to the gene of interest. Plasmid extraction should be performed using an endotoxin-free plasmid extraction kit to ensure the plasmid has an A₂₆₀/A₂₈₀ ratio of 1.8–2.0 and a concentration of no less than 500 ng/μL. Avoid repeated freeze-thaw cycles of dissolved plasmids; aliquot according to usage and store at -20°C.
(2) Add 1.8 mL of serum-free and antibiotic-free basal medium to the above mixture, mix gently, and let stand at room temperature for 30 minutes to form transfection reagent-nucleic acid complexes.
⁕ Transfection reagent-nucleic acid complexes remain stable for up to 4 hours at room temperature.
(3) Gently add the 1.8 mL transfection reagent-nucleic acid complexes dropwise and evenly to the packaging cells in the 10 cm dish. Swirl the dish gently to mix, then incubate in a 37°C, 5% CO₂ incubator for virus production.
3. Virus Harvest
(1) At 24 hours post-transfection, optionally discard the initial viral supernatant and add 10–15 mL of fresh complete medium (containing serum) to the 10 cm dish. Continue incubation in a 37°C, 5% CO₂ incubator (this step is optional).
(2) At 48 hours post-transfection, collect the viral supernatant, then add 10–15 mL of fresh complete medium (containing serum) to the 10 cm dish. Continue incubation in a 37°C, 5% CO₂ incubator.
(3) At 72 hours post-transfection, observe and photograph the cell status, then collect the viral supernatant a second time. Combine it with the supernatant collected at 48 hours, centrifuge at 800×g for 10 minutes to remove cell debris, and filter through a 0.45 μm filter. The filtered supernatant can be used directly to infect target cells or concentrated using the Lentivirus Concentration Kit (S1506239) before infection.
⁕ Avoid repeated freeze-thaw cycles of the virus, as each freeze-thaw cycle reduces the viral titer by 10%–20%. Lentiviruses stored at -80°C are best used within six months.
II. Viral Titer Determination
1. Calibration of Fluorescent Recombinant Lentiviral Titer by Well Dilution Method
(1) Day 1: Cell Preparation
Digest and count well-grown 293T cells, then dilute them to a concentration of 1–3×10⁵ cells/mL. Add the cell suspension to a 96-well plate at a volume of 100 μL per well (corresponding to 1–3×10⁴ cells per well). Set 6 gradient wells for each type of lentivirus, and incubate overnight in a 37°C, 5% CO₂ incubator.
(2) Day 2: Virus Dilution and Infection
Perform 10-fold serial dilutions in EP tubes, with 6 consecutive dilution gradients. The dilution method is as follows: Prepare 6 sterile EP tubes, and add 90 μL of fresh complete medium (containing 10 μg/mL Polybrene) to each tube. Take 10 μL of the lentivirus solution to be tested and add it to the first EP tube (labeled "10 μL"). After mixing, take 10 μL of the virus dilution and add it to the second EP tube (labeled "1 μL"). Repeat this process until the last tube is diluted. Discard the original medium in the 96-well plate, then add the diluted virus to the wells in sequence and label them properly. Operate carefully to avoid disturbing the cells, and incubate in the incubator for 16 hours. If replicate wells are required, increase the above reagent volumes proportionally according to the number of replicate wells.
(3) Day 3: Remove Virus and Replace Medium
Discard the virus-containing medium, and add 100 μL of pre-warmed fresh complete medium to each well.
(4) Day 4: Fluorescence Counting and Titer Calculation
Method 1: End-Point Dilution Counting Method
At 48 hours post-infection, count the fluorescence-positive cells using a fluorescence microscope. Count the number of fluorescent cells in the last two wells (if replicate wells are set, calculate the sum of the total number of cells in the replicate wells and then the average). Denote the counts as A (number of fluorescent cells in the penultimate well) and B (number of fluorescent cells in the last well).
Lentiviral Titer Calculation Formula 1:Viral titer (TU/mL) = (A + B × 10) × 1000 / 2 / Volume of virus in Well A (μL)
Example 1:If the number of fluorescence-positive cells corresponding to Tube 5 is 10, and that corresponding to Tube 6 is 2, the viral titer is calculated as follows:Titer (TU/mL) = (10 + 2 × 10) × 1000 / 2 / 0.001 = 1.5 × 10⁷
To infect 2 × 10⁵ cells with an MOI (Multiplicity of Infection) of 5 (5 viral particles per cell), the required virus volume is:Required virus volume = (2 × 10⁵ × 5) / (1.5 × 10⁷) (mL) = 0.067 mL = 67 μL
Method 2: Appropriate Fluorescence Counting Method
At 48 hours post-infection, use a fluorescence microscope or flow cytometry to count two consecutive gradients with a suitable fluorescence ratio (10%–50%). Count the number of fluorescent cells in these two wells (if replicate wells are set, calculate the sum of the total number of cells in the replicate wells and then the average). Denote the counts as C (number of fluorescent cells in the well with higher virus concentration) and D (number of fluorescent cells in the well with lower virus concentration).
Lentiviral Titer Calculation Formula 2:Viral titer (TU/mL) = (C + D × 10) × 1000 / 2 / Volume of virus in Well C (μL)
Example 2:If the number of fluorescence-positive cells corresponding to Tube 3 is 10,000, and that corresponding to Tube 4 is 2,000, the viral titer is calculated as follows:Titer (TU/mL) = (10,000 + 2,000 × 10) × 1000 / 2 / 0.1 = 1.5 × 10⁸
To infect 2 × 10⁵ cells with an MOI of 30 (30 viral particles per cell), the required virus volume is:Required virus volume = (2 × 10⁵ × 30) / (1.5 × 10⁸) (mL) = 0.04 mL = 40 μL
2. Calibration of Non-Fluorescent Recombinant Lentiviral Titer by Relative Quantitative PCR
(1) Day 1: Cell PreparationDigest and count well-grown 293T cells, then dilute them to a concentration of 1–3×10⁵ cells/mL. Add the cell suspension to a 24-well plate at a volume of 500 μL per well (corresponding to 0.5–1.5×10⁵ cells per well). Set 6 gradient wells for each type of lentivirus, and incubate overnight in a 37°C, 5% CO₂ incubator.
(2) Day 2: Virus Dilution and InfectionPerform 10-fold serial dilutions in EP tubes, with 6 consecutive dilution gradients. The dilution method is as follows: Prepare 6 sterile EP tubes, and add 450 μL of fresh complete medium (containing 10 μg/mL Polybrene) to each tube. Take 50 μL of the lentivirus solution to be tested and add it to the first EP tube (labeled "50 μL"). After mixing, take 50 μL of the virus dilution and add it to the second EP tube (labeled "5 μL"). Repeat this process until the last tube is diluted. Discard the original medium in the 24-well plate, then add the diluted virus to the wells in sequence and label them properly. Operate carefully to avoid disturbing the cells, and incubate in the incubator for 16 hours. If replicate wells are required, increase the above reagent volumes proportionally according to the number of replicate wells.
(3) Day 3: Remove Virus and Replace Medium
Aspirate the virus-containing medium, and add 500 μL of pre-warmed fresh complete medium to each well.
(4) Day 4: Relative Quantitative PCR and Titer Calculation
Wash the cells with PBS, then detach them by pipetting. Collect the cells by centrifugation, extract genomic DNA, and perform qPCR detection.
Use serial dilutions of the lentiviral vector plasmid (to be tested) as the standard, and perform qPCR with the universal primer RRE (targeting the lentiviral vector) to obtain the number of virus integration copies.
Use serial dilutions of the ACTB plasmid as the standard, and perform qPCR with ACTB primers to obtain the number of genomic copies.
Number of lentiviral copies per cell = 2 × Number of lentiviral copies / Number of ACTB copies
Lentiviral Titer Calculation Formula 3:Titer (TU/mL) = (E × N × 1000) / Volume of virus in the corresponding well (μL)
Where:
E = Number of lentiviral copies per cell
N = Number of cells at the time of infection (approximately 1.5 × 10⁵)
⁕ During the determination, set up a group of fluorescent lentiviruses with known TU (transducing units) as a control to verify the detected values.
III. Lentiviral Infection of Target Cells
The tropism of the VSVG envelope protein for different cells and tissues varies significantly. Before infecting target cells with lentivirus, it is necessary to consult relevant literature or conduct pre-experiments to determine the multiplicity of infection (MOI) for the target cells.
1. Package the lentivirus and determine its titer according to the above steps. Calculate the required amount of virus based on the viral titer and MOI (refer to 06/Experimental Steps/Viral Titer Determination for the calculation formula).
2. One day before the experiment, digest and count well-grown target cells, then dilute them to a concentration of 1–3×10⁵ cells/mL. Add the cell suspension to a 24-well plate at a volume of 500 μL per well (corresponding to 0.5–1.5×10⁵ cells per well). Incubate overnight in a 37°C, 5% CO₂ incubator to ensure the cell confluency reaches approximately 30%–50% when viral infection is performed the next day.
⁕ Adherent cells do not require seeding one day in advance. After counting and seeding the cells before the experiment, directly add the virus mixture.
3. Prepare a mixture of lentivirus + Polybrene + medium. Polybrene is a commonly used infection-promoting reagent that can significantly improve the contact between virus and cells as well as infection efficiency. Its general working concentration is 2–10 μg/mL; however, it exhibits high toxicity to certain cell types (such as terminally differentiated neurons, DC cells, immune cells, and stem cells). For first-time use, it is recommended to conduct a toxicity test first. Compared with the cytotoxicity caused by adding Polybrene, the Viral Transfection Enhancer (V1501911) has extremely low cytotoxicity and causes almost no damage to cell growth. Therefore, for cells that cannot use Polybrene, the Viral Transfection Enhancer (V1501911) is the preferred viral infection enhancer.
4. Aspirate the medium of target cells in the 24-well plate, add the lentivirus mixture prepared in Step 3, and continue incubation in a 37°C, 5% CO₂ incubator.
⁕ For infecting suspension cells or semi-suspension cells, the horizontal centrifugation transfection method should be used: add an appropriate amount of virus solution to the cell culture plate, seal the plate tightly, place it in a horizontal centrifuge, centrifuge at 800×g for 30 minutes (varies by cell type), and then transfer it to the incubator for continued culture.
5. Replace with fresh medium 16 hours after viral infection and continue incubation.
6. Forty-eight hours after viral infection, detect the expression effect using methods such as fluorescence detection, Western Blot, RT-PCR, ELISA, flow cytometry, or reporter gene assays according to actual conditions; alternatively, add selection drugs to screen for stable cell lines.
7. If the expression effect is not ideal, adjust the MOI (e.g., set up an MOI gradient) and perform the detection again.
Scope of Application:
It is compatible with most lentiviral expression vectors. Through design and optimized proportioning, it ensures that recombinants are packaged into lentiviral particles with high infectivity, while preventing self-replication of the virus.
Precautions:
1. When used in conjunction with the Lentivirus Packaging Cell Line, this system can package lentiviruses with higher titer and higher expression levels.
2. After harvesting the crude virus, it is recommended to aliquot an additional several small-volume tubes (20–100 µL each) and store them at -80°C together with other aliquoted large-volume virus samples. Once these small-volume virus samples are frozen, take them out to thaw and then determine the titer. This step can simulate the decrease in active titer caused by one freeze-thaw cycle, ensuring the measured titer is truly referenceable.
3. For your health and safety, please operate in compliance with standard procedures and wear a lab coat and gloves when conducting experiments.
4. All lentivirus-related operations must be performed in a BSL-2 (Biosafety Level 2) biological safety cabinet.
5. This product is for research use only and shall not be used for clinical diagnosis or treatment.
Analysis of Experimental Case:
1. One day in advance, culture the Lentivirus Packaging Cell Line in a 10 cm cell culture dish. Start lentiviral packaging when the cell confluency reaches approximately 75%.
2. Use the Lentiviral Packaging Kit (L1455942) for lentiviral packaging. Add 4 μL of the Lentiviral Packaging Helper Plasmid Mix (Package Plasmid Mix) to a 2 mL centrifuge tube, then add 4 μL of the control plasmid expressing GFP (Control Plasmid). Mix gently, add 40 μL of Lentivirus Transfection Reagent to the plasmid mixture, and incubate at room temperature for 3 minutes.
3. Add 1.8 mL of serum-free and antibiotic-free DMEM basal medium to the above mixture, mix gently, and let stand at room temperature for 30 minutes to form transfection reagent-nucleic acid complexes.
4. Gently add the 1.8 mL transfection reagent-nucleic acid complexes dropwise and evenly to the lentiviral packaging cells in the 10 cm dish. Swirl the dish gently to mix, then place it in a 37°C, 5% CO₂ incubator for virus production.
5. At 24 hours post-transfection, discard the initial viral supernatant, add 10 mL of fresh complete medium (DMEM + 10% FBS) to the 10 cm cell culture dish, and continue incubation in a 37°C, 5% CO₂ incubator.
6. At 48 hours post-transfection, collect the viral supernatant, add another 10 mL of fresh complete medium (DMEM + 10% FBS) to the 10 cm cell culture dish, and continue incubation in a 37°C, 5% CO₂ incubator.
7. At 72 hours post-transfection, observe the cell status, take photos, and collect the viral supernatant for the second time. Combine this supernatant with the one collected at 48 hours, centrifuge at 800×g for 10 minutes to remove cell debris, and filter through a 0.45 μm filter. Concentrate the virus 20-fold using Lentivirus Concentration Solution (S1506239), then resuspend the lentiviral particles in 1 mL of Lentivirus Stable Storage Solution (L1505998).
8. Determine the viral titer using the well dilution method (refer to the viral titer determination method in 06/Experimental Steps). Observe the expression of green fluorescent protein (GFP) using a fluorescence microscope, take photos for documentation, and the experimental results are shown in Figure 2.
Figure 2. Titer determination of lentiviruses (GFP, Control plasmid) packaged using the Lentiviral Packaging Kit (L1455942) via infecting 293T cells
