Lentivirus is a type of retrovirus that enables exogenous genes to integrate into the genome of host cells, achieving stable and long-term expression of exogenous genes. Compared with ordinary retroviruses, it has a higher titer and a broader host range. To package lentiviruses, lentiviral vectors carrying exogenous genes and packaging vectors (which provide helper proteins required for viral particle production) are co-transfected into packaging cells. The packaged lentiviruses are pseudoviruses—after infecting target cells, these pseudoviruses cannot infect other cells nor use host cells to produce new viral particles. The toxic genes in the lentivirus have been deleted and replaced with exogenous target genes. These pseudoviruses are secreted into the extracellular culture medium. After centrifugation to collect the supernatant, lentivirus concentration is performed. The concentrated lentivirus solution can be directly used to infect host cells. After the target gene enters the host cell, it undergoes reverse transcription and integrates into the host genome, thereby realizing the expression of the exogenous gene in the host cell (see the figure below).
Lentivirus Packaging Transfection Reagent (S1505995) is a novel transfection reagent based on cationic polymer. Its optimized formula is particularly suitable for viral packaging in lentivirus packaging cell lines (such as HEK293T and HEK293FT), and it possesses advantages including high efficiency, low toxicity, simple operation, and good reproducibility. It can significantly improve the packaging efficiency of lentiviral vectors in lentivirus packaging cell lines and produce recombinant lentiviruses with higher titers. The titer of control plasmid virus packaged using this transfection reagent can reach 10⁸ TU/mL, which features high infection efficiency and a broad infection spectrum. In addition, the unique formula of Lentivirus Packaging Transfection Reagent allows it to be directly added to the culture medium. The presence of serum does not affect transfection efficiency, which can reduce cell damage caused by serum removal. Meanwhile, there is no need to remove the transfection reagent-nucleic acid complex or replace it with fresh medium after transfection.
Product Features:
⁕ Excellent lentivirus packaging efficiency — Its optimized formula is particularly suitable for viral packaging in lentivirus packaging cell lines, demonstrating outstanding transfection efficiency and high-titer lentivirus expression levels.
⁕ Low cytotoxicity — It acts gently and can well balance high transfection efficiency and low cytotoxicity.
⁕ Simple operation — There is no need to remove the complex or replace with fresh medium after transfection.
⁕ Features definite chemical composition, serum-free property, and no animal-derived components, enabling high reproducibility and batch-to-batch reliability.
Scope of Application:
The optimized formula of this product is particularly suitable for viral packaging in lentivirus packaging cell lines (such as HEK293T and HEK293FT). It can significantly improve the packaging efficiency of lentiviral vectors in lentivirus packaging cell lines and produce recombinant lentiviruses with higher titers.
Summary of Experimental Procedure:
Figure 1. Experimental Flowchart for Lentivirus Packaging Using Lentivirus Packaging Transfection Reagent (S1505995) (The reagent amounts shown in the figure are for packaging lentivirus in a 10 cm dish).
Experimental Steps:
(Take lentivirus packaging in a 10 cm dish as an example; for the sample addition volume of other culture devices, refer to Table 1: Transfection Quantity Standards)
1. Cell Preparation
One day before transfection, seed 4–6×10⁶ lentivirus packaging cells (such as HEK293T, HEK293FT, or Lentivirus Packaging Cell Line) into a 10 cm cell culture dish, ensuring the cell density reaches 70%–80% at the time of packaging. Incubate the dish in a 37°C, 5% CO₂ incubator for 16–24 hours.
⁕ Cell status significantly affects viral packaging efficiency. Cells to be used for packaging should be in good growth condition. It is recommended to use cells in the exponential growth phase with a survival rate >90% for lentivirus packaging.
⁕ When used in combination with the Lentivirus Packaging Cell Line, this transfection reagent can produce lentiviruses with higher titers and higher expression levels.
2. Lentivirus Packaging
(1) Add 4 μL of Package Plasmid Mix to a 2 mL centrifuge tube, then add 4 μg of lentiviral vector plasmid containing the target gene. Mix gently, add 40 μL of transfection reagent to the plasmid mixture, and incubate at room temperature for 3 minutes.
⁕ The lentiviral vector plasmid needs to be constructed according to the gene of interest. Plasmid extraction should be performed using an endotoxin-free plasmid extraction kit to ensure the A₂₆₀/A₂₈₀ ratio of the plasmid is 1.8–2.0 and the concentration is no less than 500 ng/μL. Avoid repeated freeze-thaw cycles of the dissolved plasmid; aliquot according to usage and store at -20°C.
(2) Add 1.8 mL of serum-free basal medium to the above mixture, mix gently, and let stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
⁕ The transfection reagent-nucleic acid complex remains stable at room temperature for up to 4 hours.
(3) Gently add the 1.8 mL transfection reagent-nucleic acid complex dropwise and evenly to the lentivirus 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.
3. Virus Harvest
(1) At 24 hours post-transfection, optionally discard the initial viral supernatant, add 10–15 mL of fresh complete medium (containing serum) to the 10 cm cell culture dish, and 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 cell culture dish and continue incubation in the 37°C, 5% CO₂ incubator.
(3) At 72 hours post-transfection, observe and photograph the cell status, then collect the viral supernatant for the 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 Lentivirus Concentration Reagent (5×) (S1506239) before infection.
⁕ Avoid repeated freeze-thaw cycles of the virus, as each freeze-thaw cycle will reduce the viral titer by 10%–20%. Lentiviruses stored at -80°C are best used within six months.
Table 1: Transfection Volume Standards
Specifications of Cell Culture Equipment | Culture System (mL)
| Total DNA (μg)
| Transfection Reagent (μL)
| Volume of Serum-Free Basal Medium (μL)
|
6-well plate
| 2 | 2 | 8
| 400
|
60mm Dish
| 4 | 4 | 8-16
| 800
|
100mm Dish
| 10 | 10 | 20-40
| 1800
|
125mL Shake Flask
| 30-35
| 30-35
| 60-120
| 6000
|
500mL Shake Flask
| 120-140
| 120-140
| 250-500
| 25000
|
1000mL Shake Flask
| 240-280
| 240-280
| 500-1000
| 50000
|
Precautions:
1. Nucleic Acid Quality: To achieve the highest transfection efficiency and lowest cytotoxicity, high-quality nucleic acids with high purity, sterility, no contamination, and no endotoxins should be used. Endotoxins in plasmids are a major obstacle to transfection, as they can significantly reduce transfection efficiency—especially for endotoxin-sensitive cells such as primary cells, suspension cells, and hematopoietic cells. It is recommended to use an endotoxin-free plasmid extraction kit for plasmid isolation to ensure the plasmid has an A₂₆₀/A₂₈₀ ratio of 1.8–2.0. Meanwhile, the amount of plasmid used should be calculated appropriately; excessive plasmid for transfection may cause cytotoxicity or even cell death.
2. Packaging Cell Quality: Cell status greatly affects transfection efficiency, which in turn impacts lentivirus quality. It is recommended to use cells in the exponential growth phase with a survival rate >90% for lentivirus packaging. When used in combination with the Lentivirus Packaging Cell Line, this transfection reagent can produce lentiviruses with higher titers and higher expression levels.
3. Cell Density: Lentivirus packaging is recommended to be performed within 12–24 hours after cell passage, when the cell density is approximately 70%–80%. Ensure consistent seeding conditions during the experiment to guarantee the reproducibility of experimental data.
4. Dosages of Plasmid and Transfection Reagent: For the recommended dosages of total DNA (μg) and transfection reagent (μL), refer to Table 1: Transfection Quantity Standards. To obtain transfection conditions more suitable for your lentivirus packaging system, further optimization can be conducted based on the recommended dosages.
5. The Lentivirus Packaging Transfection Reagent can be used for transfection in serum-containing medium, and there is no need to change the medium before or after transfection. However, when preparing the transfection complex, serum-free basal medium is required to dilute the DNA and transfection reagent, as serum may interfere with the formation of the complex.
6. All lentivirus-related operations must be performed in a Biosafety Level 2 (BSL2) biological safety cabinet.
7. For your health and safety, please operate in a standardized manner and wear a lab coat and gloves during the experiment.
8. This product is for research use only and shall not be used for clinical diagnosis or treatment.
Experimental Case Analysis:
1. One day in advance, culture the Lentivirus Packaging Cell Line in a 10 cm cell culture dish. Start lentivirus packaging when the cell density reaches approximately 75%.
2. Add 4 μL of the Lentivirus Packaging Helper Plasmid Mix (Package Plasmid Mix, Cat. No. L1455942) to a 2 mL centrifuge tube, then add 4 μL of the GFP-expressing control plasmid (Control Plasmid). Mix gently, add 40 μL of the transfection reagent (Cat. No. S1505995) to the plasmid mixture, and incubate at room temperature for 3 minutes.
3. Add 1.8 mL of serum-free DMEM basal medium to the above mixture, mix gently, and let it stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
4. Gently add the 1.8 mL transfection reagent-nucleic acid complex dropwise and evenly to the lentivirus packaging cells. Swirl the culture 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 the 37°C, 5% CO₂ incubator.
6. At 48 hours post-transfection, collect the viral supernatant, then add 10 mL of fresh complete medium (DMEM + 10% FBS) to the 10 cm cell culture dish and continue incubation in the 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 filtrate 10-fold using Lentivirus Concentration Solution (5×) (Cat. No. S1506239), then resuspend the lentiviral particles in 2 mL of Lentivirus Stable Storage Solution (Cat. No. L1505998).
8. Determine the viral titer using the serial dilution method (well dilution method). Observe the expression of green fluorescent protein (GFP) under a fluorescence microscope, take photos for documentation, and the experimental results are shown in Figure 2.
Figure 2. Titer Detection of Lentivirus (GFP) Packaged Using Lentivirus Packaging Transfection Reagent (S1505995), with GFP (Green Fluorescent Protein) Expression Detected by Fluorescence Microscopy.
