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Generating Stable Cell Lines with Lentivirus
Section.01
Why Choose Lentivirus for Stable Cell Line Development?

Derived from HIV-1, lentiviral vectors are widely favored for stable cell line development due to their ability to stably integrate transgenes into the host genome. This enables long-term, consistent gene expression across multiple cell divisions, in contrast to the transient expression achieved with standard transfection methods. Consequently, experiments conducted with lentivirus-generated stable cell lines exhibit superior reproducibility by eliminating inter-experimental variability inherent to repeated transient transfections.

Table 1. Key Features of Different Viral Vectors

Notes on Viral Titer Units:

TU/mL (Transducing Units/mL): Used for lentiviral vectors, represrmting the concentration of functional viral particles capable of transducing target cells and expressing the transgene.

PFU/mL (Plaque Forming Units/mL): Applied to adenoviruses vectors, quantifying the number of infectiousviral particles based on plaque formation in cell cultures.

VG/mL (Viral Genomes/mL): Used for AAV vectors, quantifying the total number of viral genomes (including both functional and non-functional particles) -which does notdirectly correlating to transduction efficiency.

Section.02
Step-by-Step Protocol for Generating Lentiviral Stable Cell Lines

Step 2. The Process of Lentivirus Packaging and Harvesting

1.Selecting Functional Elements for Efficient Selection & Tracking.

①Eukaryotic Resistance Genes (e.g., Puromycin/Blasticidin): Enable effective antibiotic selection, eliminating non-transduced cells.

②Fluorescent Reporters (e.g., GFP/mCherry): Enable real-time visualization of transduction efficiency and expression.

③Epitope Tags (e.g., HA/Flag): Facilitate detection and validation through techniques such as Western blot (WB) or immunoprecipitation (IP).

2.Choosing the Right Vector Based on Experimental Goals

①For Gene Overexpression: Vectors such as pSLenti-CMV-EGFP-3xFLAG-WPRE enable robust and sustained expression.

②For Gene Knockdown: Select constructs like pCLenti-U6-shRNA-CMV-Puro-WPRE for effective and stable target silencing.

Technical Recommendations:

· If the gene of interest exceeds 4 kb, perform a small-scale test to evaluate packaging efficiency, as large inserts may significantly reduce viral titer.

· Establish an appropriate selection concentration using a kill curve assay before formal selection to avoid excessive cell death.

· Loss of reporter signal over serial passages may indicate transgene silencing or genomic instability; consider early cryopreservation of validated pools or clones.

· If an epitope tag is structurally masked, introduce flexible linkers (e.g., (G4S)×3) to improve accessibility for antibody binding.

Figure 1. Schematic diagram of a lentiviral vector.

Step 2. The Process of Lentivirus Packaging and Harvesting

Figure 2. The process of lentivirus packaging and harvesting.

1.Prepare cells

24 h prior to transfection, plate 4-6×106 of cells (293 or 293T, etc.) on a 10 cm dish and incubate until the density reaches 70%-80% for cell transfection.

2.Lentivirus packaging

(1) Take 4 μL of lentivirus packaging helper plasmid mixture into a 2 mL centrifuge tube, add 4 μg of lentiviral vector plasmid containing the target gene, mix gently, add 40 μL of MCE Lentivirus Transfection Reagent and mix gently, incubate for 3 min at room temperature.

(2) Add 1.8 mL of serum-free basal medium, mix gently and incubate for 30 min at room temperature.

Note: Transfection reagent-nucleic acid complex is stable for 4 h at room temperature.

(3) Add the Transfection reagent-nucleic acid complexes (1.8mL) to the cells and mix gently, and incubator for further culture.

Note: Take care to be slow in dripping to avoid washing up the cells and to mix gently.

3.Harvesting of Lentivirus

(1) After transfection for 24 h, discard the initial viral supernatant, add 10-15 mL of fresh complete medium (including serum), and continue incubating for further culture.

(2) After transfection for 48 h, collect the viral supernatant, add 10-15 mL of fresh complete medium (including serum), and continue incubating further culture.

(3) After transfection for 72 h, observe the cell state and take pictures. Collect the viral supernatant, mix with the supernatant collected by 48 h. Centrifuge the mixture at 800 × g for 10 min to remove cell debris, then filter it using a 0.45 μm filter. The filtered supernatant can be used to directly infect the cells or concentrated to obtain a lentiviral concentrate with higher titer.

Note: The titer of the virus will be reduced by 10%-20% within a freeze-thaw. Lentivirus is recommended to be stored at -80°C after dispensing and used within six months.

Step 3. Lentiviral Transduction of Target Cells

Note: The following procedure uses puromycin selection as an example.

The optimal multiplicity of infection (MOI) varies across cell types and must be determined empirically. MOI refers to the ratio of viral particles to target cells during transduction. An MOI yielding approximately 80% transduction efficiency is generally recommended for stable cell line generation.

Figure 3. Schematic representation of generation of stable cell lines using lentivirus.

1. Determining Minimum Lethal Concentration of Puromycin (Kill Curve Assay)

(1) Plate the target cells into a 24-well plate.

(2) Prepare a puromycin stock solution (1 mg/mL) and add to the cells in gradient concentrations as indicated in the reference table.

Table 2. Example calculation of puromycin stock solution volumes for preparing gradient puromycin concentrations.

(3) After 48 hours, identify the lowest dose that completely kills all of the cells. This will be the working puromycin concentration for selection.

2. Identifying Optimal MOI for Target Cells

(1) One day before transduction, seed 5,000–10,000 cells per well of a 96-well plate in 100 µL medium, targeting ~40% confluency at plating and ~70% at the time of transduction.

(2) Thaw the lentiviral stock on ice. Dilute an appropriate aliquot in PBS or serum-free medium to 1×10⁸ TU/mL. Store the remaining virus at 4°C.

(3) Cell Transduction

a)Calculate the required virus volume using the formula:Virus volume (µL) = (MOI × Cell number) / Viral titer × 1000

b)Refer to Table 2 for sample calculations based on MOI values of 1, 10, 20, 50, and 100, with a viral titer of 1×10⁸ TU/mL.

Table 3. Recommended Parameters for Cell Transduction (Viral Titer: 1E+8 TU/mL)

c)Add the calculated virus volume to each well, mix gently, and incubate overnight at 37°C with 5% CO₂.

(4) Selection and Evaluation: After 24–48 hours, replace the medium with fresh medium containing the predetermined puromycin concentration. Continue selection for 3–5 days, refreshing the medium as needed. Under microscopy, identify wells with high cell survival and healthy morphology as indicating the suitable MOI.

3. Formal Experiment: Stable Cell Line Selection

Once the optimal puromycin concentration and MOI are determined, scale up the transduction while maintaining the initial cell seeding density and proportionally adjusting medium and virus volumes.

(1) Plate cells at a density that will reach ~50% confluency the next day. Incubate overnight at 37°C.

(2) Thaw the lentivirus on ice and dilute to the desired concentration using PBS or serum-free medium. Mix gently by pipetting.

(3) Replace the cell culture medium, then add the diluted virus dropwise. Swirl gently and incubate overnight.

(4) At 24 hours post-transduction, dissociate the transduced cells with Trypsin and replate them at a low density to facilitate optimal growth and selection. After 3 hours, add puromycin to begin selection.

Refresh the medium the next day and passage every other day under maintained puromycin pressure.

(5) Isolate and expand single-cell colonies under continued puromycin selection. Validate target gene expression via Western blot, PCR, or other appropriate methods. Select well-validated stable cell lines for cryopreservation.

Section.03
MedChemExpress Lentivirus Packaging Services

MedChemExpress (MCE) provides a comprehensive portfolio of high-titer lentiviral products and services to support both coding and non-coding gene studies. Our lentiviruses are produced under rigorous quality control using multi-plasmid systems in 293T cells, followed by purification via ultracentrifugation. Each batch is validated to deliver titers ≥1×10⁸ TU/mL and is certified free of mycoplasma, endotoxins, and other contaminants—ready to power your next discovery.

Let MCE’s trusted tools accelerate your stable cell line development!