Lentiviral Packaging Protocol

For Reference Only; Optimization May Be Required Depending on Laboratory Conditions

I. Lentiviral Packaging

1. Overview

The lentiviral packaging system consists of a packaging plasmid mixture and a lentiviral transfer vector. The lentiviral vector plasmid pLVX-Puro contains the essential HIV elements, including the 5′ LTR and 3′ LTR, as well as additional accessory elements.

The pSPAX2 plasmid contains the gag gene, which encodes the major structural proteins of HIV, and the pol gene, which encodes viral-specific enzymes.

The pMD2.G plasmid contains the VSV-G gene, which provides the envelope protein required for lentiviral packaging.

2. Materials

• 293T cells in the exponential growth phase

• Transfection reagent

• Plasmid mixture: pLVX : pSPAX2 : pMD2.G = 4 : 3 : 1

All of the above plasmids are available from the MiaolingBio platform.

3. Packaging Procedure

① Cell seeding:
Digest and collect the cells using trypsin. Seed the cells into an appropriate culture dish according to your experimental requirements and incubate at 37°C with 5% CO₂ for 8–24 hours. Perform transfection after the cells have attached.

② Complex preparation:
Thoroughly mix the transfer vector, packaging plasmids, and transfection reagent, then allow the mixture to stand for 15–20 minutes.

③ Transfection:
Add the mixture dropwise to the cell culture dish. Gently mix and incubate at 37°C with 5% CO₂.

④ Virus harvest:
Collect the lentivirus-containing culture supernatant at approximately 24 and 48 hours after transfection. Aliquot and store the viral supernatant at −80°C. If the viral titer is relatively low, the virus can be concentrated by ultracentrifugation.

⑤ Target-cell transduction:
Seed the target cells into an appropriate culture dish. Replace the culture medium after 12–24 hours. A cell density of approximately 30–50% confluence is recommended. Add the collected viral supernatant and continue culturing the cells. Once the cells reach confluence, passage the cells or assess transgene expression as needed.

II. Lentiviral Titer Determination

1. For viral vectors containing a fluorescent marker or reporter gene, the viral titer can be determined by transducing cells and measuring reporter expression.

2. For viral vectors without a reporter gene, the viral titer can be estimated using an appropriate assay kit to measure the activity or concentration of viral proteins associated with viral particles.

3. Viral titer can also be determined by quantitative PCR (qPCR).

III. Precautions

1. Avoid using plasmids prepared by miniprep when possible. Miniprep DNA generally has lower concentration and purity than plasmids prepared by maxiprep, which may reduce lentiviral packaging efficiency.

2. Maintain an appropriate cell density during transfection. A cell density of approximately 40–60% confluence is generally suitable. If the cells become overly confluent, promptly replace or supplement the culture medium with fresh medium to maintain healthy cell growth.

3. Clarify the viral supernatant after collection. Centrifugation can remove cellular debris and a small number of suspended 293T cells. If necessary, filtration can be performed to further remove cellular contaminants.

4. Consider filtration during virus collection. Filtration can help remove cellular contaminants and residual VSV-G-associated components that may be cytotoxic to cells, although filtration may also result in some loss of viral titer.

5. Avoid repeated freeze-thaw cycles. Repeated freezing and thawing can reduce viral infectivity. Storing lentiviral preparations at 4°C for extended periods may also decrease infectivity.

6. VSV-G-mediated cytotoxicity: VSV-G mediates membrane fusion between viral particles and cells and may cause cytotoxicity in some cell types. Therefore, the volume used to resuspend concentrated viral particles should be determined based on the specific experimental requirements.

7. Target-cell density is critical for transduction efficiency. Cell density can significantly affect lentiviral transduction efficiency. Excessively high cell density or over-confluent cultures may reduce transduction efficiency.