The target cells can be transduced by AAV, but why is the GFP fluorescence intensity very low?

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.

The transduction efficiency of AAV in target cells is low. How can the virus transduction efficiency be improved?

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.

What is the packaging capacity of AAV viruses?

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.

How long does the lentivirus packaging process take, and what affects the turnaround time?

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 modificationsAdditional quality control steps requested by the customer

Why do cells not grow or even die after lentiviral infection?

（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.

Why does the company validate effective lentivirus, but it shows no expected effect on the target cells?

（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.

What is included in the adenovirus packaging service?

Our service provides high-titer, high-purity adenoviral vectors customized to your needs. Each delivery includes target and control viruses, precise titers, a detailed shipping list, and usage instructions for seamless integration into your experiments.

What makes adenovirus different from other viral vectors?

Adenovirus enables rapid, strong, and transient gene expression in a broad range of mammalian cells. It remains episomal without integrating into the host genome, supports large genes, and provides high transduction efficiency, making it ideal for in vitro and in vivo studies.

What is the difference between Viral Particles (VP), Plaque Forming Units (PFU), and Infectious Units (IFU)?

Viral Particles (VP) represent the total number of both live and dead viral particles and do not reflect the amount of active virus in the preparation. Plaque Forming Units (PFU) represent the number of infectious or live viruses, providing a measure of the active virus present. Infectious Units (IFU) are equivalent to PFU. For most virus preparations, the VP/PFU ratio is 20:1 to 50:1. Using VP as a measurement can result in significant variations in the amount of actual live viruses present, whereas using IFU or PFU gives more consistent outcomes.

How to assess the quality of a mutation library?

Key indicators for assessing the quality of a mutation library include:- Diversity of mutations: The mutations in the library should cover a wide range of variations in the target gene, ensuring sufficient representativeness.- Complexity of the library: This refers to the number of mutant variants in the library, ensuring the library size is large enough to cover possible mutation combinations.- Accuracy of mutations: The accuracy of mutations in the library should be verified using high-throughput sequencing techniques (such as NGS) to avoid the introduction of erroneous mutations.

How to choose the right virus titer for my experiment?

- Consider the target cell type, as some cells are more sensitive than others.- Higher titers aren't always better—too high can be toxic or trigger immune responses.- Start with small-scale tests to determine the optimal functional titer.

How to verify successful gene knockdown or activation after using a library?

- Use qPCR or Western blotting to check gene expression changes.- Functional assays (like reporter assays or phenotypic readouts) can confirm the library's effect.- Sequencing can confirm the guide RNA or mutation was correctly introduced.

What are polyclonal stable cell lines and monoclonal stable cell lines?

Polyclonal stable cell line (cell pool): This is obtained by directly selecting cells with drug resistance after transfection. It consists of multiple clones from different sources, with varying integration sites and expression levels of the exogenous gene. It has a short production cycle and low cost, suitable for initial screening and high-expression applications.Monoclonal stable cell line: This is derived from a cell pool through methods such as limiting dilution, and consists of a single clone of cells with stable integration of the exogenous fragment. All cells have the same integration site, ensuring consistency and stability in expression. It is suitable for high-precision research and biopharmaceutical production. However, it has a longer production cycle and higher cost, and is ideal for cell line experiments requiring subcellular localization, difficult-to-infect cells, and low-expression applications.

What validation data will I receive with the constructed cell line?

Each project includes comprehensive validation such as sequencing results, qPCR data, and optional protein-level verification (e.g., Western blot), ensuring reliable and reproducible results.

What are the advantages and disadvantages of overexpression vs. gene knock-in?

Overexpression cell lines are faster and more cost-effective to construct, and they are suitable for studying gene function at the protein level. However, expression may be non-physiological due to strong promoters or random genomic integration. Knock-in cell lines, on the other hand, place the gene under its natural promoter, allowing physiologically relevant expression and more accurate functional studies. The trade-off is that knock-in projects are more complex and typically require longer timelines.

Gene Delivery - Beijing Tsingke Biotech Co., Ltd.

Q: What are the common methods for gene overexpression?

Common methods for gene overexpression:Overexpression plasmids: Introduce the target gene into cells through plasmid transfection for overexpression.Overexpression lentivirus: Use lentiviral vectors to stably transduce the target gene, suitable for long-term gene expression.CRISPRa transcriptional activation system: Utilize CRISPR technology to activate the expression of endogenous genes, offering precise regulation.Each method has its own characteristics, and the choice should be based on the experimental objectives and cell types.

Q: How long does it take to receive packaged viral vectors?

Turnaround time varies by vector type and project scale. Typically, AAV, LV, and AdV vectors can be delivered in as fast as 12–18 business days. Larger-scale or customized projects may take longer.

Q: Can I get a guarantee service for virus packaging?

Yes. For each category of viruses (AAV, ADV, lentivirus, etc.), clients can opt for a guarantee service to ensure the viral vectors meet expected performance standards.

Q: Can you help me generate stable cell lines?

Yes. We offer custom stable cell line construction, including knockout, overexpression/interference, point mutation, and knock-in cell lines.Delivered as frozen or cultured cells (≥1×10⁶ cells per vial)Turnaround time: 6–11 weeks depending on cell typeQuality verification: Sanger sequencing, qPCR, or protein-level validationIncludes positive clones and, when applicable, control lines ready for downstream experiments

How to choose between AAV, LV, and ADV for different viral vector applications?

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Adeno-associated virus(AAV):Suitable for long-term gene expression and applications with high safety requirements (e.g., gene therapy).

Advantages: Long-term expression, high safety.

Disadvantages: Lower transduction efficiency, small gene capacity.

Lentivirus(LV):Suitable for stable transduction and long-term gene expression (e.g., cell therapy).

Advantages: High transduction efficiency, broad cell type applicability, long-term expression.

Disadvantages: Potential genome integration, safety risks.

Adenovirus (ADV):Suitable for short-term expression and large-scale cell transduction (e.g., vaccine development).

Advantages: High transduction efficiency, suitable for large-scale production.

Disadvantages: Short-term expression, can trigger immune response.

Choose the appropriate vector based on your experimental needs.

What are the common methods for gene overexpression?

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How long does it take to receive packaged viral vectors?

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Do I need to prepare my own plasmids?1.No. Our service includes plasmid preparation, where we clone the gene or sgRNA cassette into an appropriate transfer plasmid and prepare high-quality DNA.

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Can I get a guarantee service for virus packaging?

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Can you help me generate stable cell lines?

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Explore the power of the Gene Factory and discover how Tsingke's integrated platform accelerates your R&D and product development.

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2025-07-31

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