慢病毒是一种逆转录病毒，能使外源基因整合入宿主细胞的基因组，实现外源基因稳定、长期的表达，比一般的逆转录病毒具有更高的滴度和更广的宿主范围。携带外源基因的慢病毒载体与包装载体(产生病毒颗粒所需的辅助蛋白)一同转染包装细胞，即可进行病毒的包装。包装好的慢病毒为假型病毒(病毒感染目的细胞后不会再感染其他细胞，也不会利用宿主细胞产生新的病毒颗粒，慢病毒中的毒性基因已被剔除并被外源性目的基因所取代)，分泌到细胞外的培养基中，经过离心取得上清液后进行慢病毒浓缩，浓缩后的慢病毒液可以直接用于宿主细胞的感染，目的基因进入到宿主细胞后，经过反转录，整合到基因组，从而实现外源基因在宿主细胞中的表达（下图）。
Lentivirus Packaging Transfection Reagent (S1505995) 是一种基于阳离子高分子聚合物的新型转染试剂，优化的配方特别适合于慢病毒包装细胞系(HEK293T、HEK293FT等)的病毒包装，具有高效低毒、操作简单、重复性好等优点，能显著提高慢病毒载体在慢病毒包装细胞系中的包装效率，并能产生更高滴度的重组慢病毒，经该转染试剂包装获得的对照质粒病毒滴度可达108 TU/mL，具有感染效率高、感染谱广等特点。此外，Lentivirus Packaging Transfection Reagent 独特的配方使其可直接加入培养基中，血清的存在不会影响转染效率，这样可以减少去除血清对细胞造成的损伤。同时，转染后不需要除去转染试剂-核酸复合物或更换新鲜培养基。
产品特点：
⁕ 卓越的慢病毒包装效率——优化的配方特别适合于慢病毒包装细胞系的病毒包装，表现出卓越的转染效率和高滴度慢病毒表达水平。
⁕ 细胞毒性低——作用温和，能较好地实现高转染效率与低细胞毒性之间的平衡。
⁕ 操作简单——转染后无需去除复合物或更换新鲜培养基。
⁕ 具有化学成分明确、无血清、无动物源成分等特点，可获得高度重现性和批间可靠性。
适用范围：
该产品优化的配方特别适合于慢病毒包装细胞系(HEK293T、HEK293FT等)的病毒包装，能显著提高慢病毒载体在慢病毒包装细胞系中的包装效率，并能产生更高滴度的重组慢病毒。
实验流程概要：
图1. 采用 Lentivirus Packaging Transfection Reagent (S1505995) 进行慢病毒包装实验流程图(图中所示为包装10 cm皿的慢病毒所需试剂量)。
实验步骤：
（以10cm皿包装慢病毒为例，其他培养装置加样体积参考表一：转染量度标准）
1. 细胞准备
转染前一天，将4-6×10⁶ 个慢病毒包装细胞(HEK293T、HEK293FT、Lentivirus Packaging Cell Line 等)接种到10 cm细胞培养皿中，使其在包装时密度为70%-80%，放置于37℃，5% CO₂培养箱培养16-24小时。
⁕ 细胞状态会极大影响病毒包装效率，待包装细胞应处于良好生长状态，建议使用生长处于指数期、存活率大于90% 的细胞进行慢病毒包装。
⁕ 该转染试剂与 Lentivirus Packaging Cell Line 配合使用，可包装出更高滴度、更高表达水平的慢病毒。
2. 慢病毒包装
(1) 取4 μL慢病毒包装辅助质粒混合物(Package Plasmid Mix)加入到2 mL离心管中，加入4 μg含有目的基因的慢病毒载体质粒，轻轻混合，加入40 μL转染试剂与质粒混合，室温孵育3分钟。
⁕ 慢病毒载体质粒需根据研究基因自行构建，应使用无内毒素质粒抽提试剂盒进行质粒提取，保证质粒A₂₆₀/A₂₈₀比值为1.8-2.0，浓度不低于500 ng/μL。溶解后的质粒切忌反复冻融，需按照使用量进行分装并于-20℃保存。
(2) 往上述混合物中加入1.8 mL无血清基础培养基，轻轻混匀，在室温下静置30分钟，形成转染试剂-核酸复合物。
⁕ 转染试剂-核酸复合物在室温下4个小时内保持稳定。
(3) 将上述1.8 mL转染试剂-核酸复合物轻轻地逐滴均匀加入10 cm皿慢病毒包装细胞中，轻轻摇动培养皿混匀，置于37℃，5% CO₂培养箱中培养产毒。
3. 收获病毒
(1) 转染24 小时后，可选择弃去初始病毒上清液，加入10-15 mL新鲜的完全培养基(含血清)至10 cm细胞培养皿中，置于37℃，5% CO₂培养箱继续培养(此步骤可选)。
(2) 转染48 小时后，收集病毒上清液，再加入10-15 mL新鲜的完全培养基(含血清)至10 cm细胞培养皿中，置于37℃，5% CO₂培养箱继续培养。
(3) 转染72 小时后，观察细胞状态并拍照，进行二次病毒上清液收集，与48 小时收集的上清液混合，800xg离心10分钟以除去细胞碎片，使用0.45 μm滤器过滤，过滤后的上清液可直接感染目的细胞，或使用慢病毒浓缩试剂 (5x)（S1506239）浓缩后感染目的细胞。
⁕ 切忌反复冻融病毒，冻融一次病毒滴度将降低10-20%。-80℃保存的慢病毒最好在半年内使用。
表一：转染量度标准
细胞培养装置规格 | 培养体系 (mL)
| Total DNA (μg)
| 转染试剂 (μL)
| 无血清基础培养基体积 (μL)
|
6孔板
| 2 | 2 | 8
| 400
|
60mm 皿
| 4 | 4 | 8-16
| 800
|
100mm 皿
| 10 | 10 | 20-40
| 1800
|
125mL 摇瓶
| 30-35
| 30-35
| 60-120
| 6000
|
500mL 摇瓶
| 120-140
| 120-140
| 250-500
| 25000
|
1000mL 摇瓶
| 240-280
| 240-280
| 500-1000
| 50000
|
注意事项：
1. 核酸质量：想要获得最高的转染效率和最低的细胞毒性，应选用高纯度、无菌、无污染、无内毒素的优质核酸。质粒中的内毒素是转染的大敌，内毒素会导致转染效率显著下降，特别是对内毒素敏感的细胞，例如原代细胞、悬浮细胞、造血细胞等。推荐使用无内毒素质粒抽提试剂盒进行质粒提取，保证质粒A₂₆₀/A₂₈₀比值为1.8-2.0。同时，需合理计算质粒用量，转染过量的质粒可能会导致细胞毒性甚至死亡。
2. 包装细胞质量：细胞状态会极大影响转染效率，从而影响慢病毒质量，建议使用生长处于指数期、存活率大于90%的细胞进行慢病毒包装。该转染试剂与 Lentivirus Packaging Cell Line 配合使用，可包装出更高滴度、更高表达水平的慢病毒。
3. 细胞密度：建议细胞传代后12-24 小时内、细胞密度约为70%-80%时进行慢病毒包装。在实验过程中保证相同的接种条件，确保实验数据的可重复性。
4. 质粒与转染试剂的用量：DNA(total μg)与转染试剂(μL)的推荐用量可以参考表一：转染量度标准。想要获得更适合您慢病毒包装体系的转染条件，可以根据推荐用量进行进一步的优化。
5. Lentivirus Packaging Transfection Reagent 可用于有血清培养基的转染，并且转染前后不需要换培养基。但是，制备转染复合物时要求用无血清基础培养基稀释DNA和转染试剂，因为血清会影响复合物的形成。
6. 所有慢病毒操作均需在BSL2级生物安全柜中进行。
7. 为了您的健康安全，请规范操作，穿戴实验服与手套开展实验。
8. 本产品仅供科研使用，请勿用于临床诊断及治疗。
实验案例分析：
1.提前一天使用10 cm细胞培养皿培养 Lentivirus Packaging Cell Line，待细胞密度为75% 左右开始包装慢病毒。
2.取4 μL慢病毒包装辅助质粒混合物(Package Plasmid Mix，L1455942)加入到2 mL离心管中，加入4 μL表达GFP 的对照质粒(Control Plasmid)，轻轻混合，加入40 μL转染试剂(S1505995)与质粒混合，室温孵育3分钟。
3. 往上述混合物中加入1.8 mL无血清DMEM基础培养基，轻轻混匀，在室温下静置30分钟，形成转染试剂-核酸复合物。
4. 将上述1.8 mL转染试剂-核酸复合物轻轻地逐滴均匀加入慢病毒包装细胞中，轻轻摇动培养皿混匀，置于37℃，5% CO₂培养箱中培养产毒。
5. 转染24 小时后，弃去初始病毒上清液，加入10 mL新鲜的完全培养基(DMEM +10% FBS)至10 cm细胞培养皿中，置于37℃，5% CO₂培养箱继续培养。
6. 转染48 小时后，收集病毒上清液，再加入10 mL新鲜的完全培养基(DMEM +10% FBS)至10 cm细胞培养皿中，置于37℃，5% CO₂培养箱继续培养。
7. 转染72 小时后，观察细胞状态并拍照，进行二次病毒上清液收集，与48 小时收集的上清液混合后，800xg离心10分钟以除去细胞碎片，0.45 μm滤器过滤，使用Lentivirus Concentration Solution (5x) (S1506239) 进行10倍浓缩，使用2 mL Lentivirus Stable Storage Solution（L1505998）重悬慢病毒颗粒。
8. 孔稀释法测定病毒滴度，使用荧光显微镜观察绿色荧光蛋白表达情况，并拍照记录，实验结果如图2所示。
图2. 采用 Lentivirus Packaging Transfection Reagent (S1505995) 包装慢病毒(GFP)进行滴度检测，采用荧光显微镜检测GFP绿色荧光蛋白表达情况。
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.
ZJ25F1230142
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