Frequently Asked Questions

The GFP fluorescence intensity in target cells depends on several factors, including the number of viral particles transducing the cells, the proliferative state of the cells, the cell type, and the observation time. Generally, the higher the number of viral particles transducing the target cells, and the faster the cell proliferation, the stronger the GFP fluorescence. For cells with faster proliferation, GFP expression typically peaks 48 to 72 hours after viral transduction. For slower proliferating cells, the GFP expression time will be extended.

At Tsingke, we generally improve transduction efficiency by increasing the MOI (multiplicity of infection). Additionally, maintaining the target cells in good growth condition is crucial to ensure normal transduction efficiency.

rAAV can package approximately 3.0 kb of genetic material. However, if you want to add a marker, such as GFP or RFP, the size of the gene that can be inserted will be smaller, around 1.5 kb.

Using your existing plasmid: The virus can be packaged and delivered in approximately 12–15 days.

Including plasmid construction: If we also need to construct the plasmid for your gene of interest, the full process typically takes 15–25 days, depending on the gene size, complexity, and any special design requirements.

Factors that can affect timing:

• Gene length (genes over 2 kb may require extra time)

• Sequence complexity or modifications

Additional quality control steps requested by the customer

（1）The most likely reason is: When lentivirus is used, an excessive viral dose can severely damage the cell genome and cell membrane. Therefore, it is necessary to determine the optimal MOI (multiplicity of infection) for each cell type, count the cells based on this value, and then use the appropriate lentiviral dose for the target cell infection.

（2）Other possible reasons: Lentiviral infection itself may induce cell differentiation, which affects the cell's proliferative ability.

（3）If the control lentiviral infection produces normal results but the target lentivirus shows abnormal results, it is possible that the gene targeted by the lentivirus is involved in processes such as the cell cycle, cell proliferation, cell metabolism, or cell adhesion. This situation is difficult to avoid but can generally be addressed by reducing the lentiviral dose, using an inducible expression system, or altering culture conditions.

（1）Functional effectiveness means: Validation is carried out on model cells (e.g., 293T, 3T3 cells) to confirm effectiveness.

（2）For overexpression and related lentiviruses, the expression of the promoter varies across different cell types. As a result, the same lentivirus infecting different cells may produce different effects.

（3）For RNA interference lentiviruses, the background expression levels of the target gene and the differences in the intracellular environment across different cell types may lead to different effects from the same lentivirus infecting different cells.