Troubleshooting the lack of surface expression of a chimeric antigen receptor (CAR) in HEK293T cells after lentiviral transduction can involve several steps. Here are some strategies to consider:

1. Check Transduction Efficiency

• Lentiviral Titer: Ensure that the lentiviral preparation is concentrated and has a high titer. Use a viral titer assay to quantify the amount of infectious virus.

• Transduction Conditions: Optimize the multiplicity of infection (MOI). A higher MOI may be necessary to achieve sufficient CAR expression.

• Transduction Time: Extend the incubation time with the virus to allow for better uptake.

2. Optimize Transduction Protocol

• Polyethylene Glycol (PEG): Consider using PEG to enhance transduction efficiency, as it can facilitate viral entry into cells.

• Centrifugation: Spinoculation (centrifuging cells with the virus) may improve transduction rates.

3. Verify CAR Expression at the mRNA Level

• RT-qPCR: Perform reverse transcription quantitative PCR to check if the CAR mRNA is being expressed in transduced cells. This will confirm whether the issue lies in transcription/translation or in surface expression.

4. Check Protein Expression

• Western Blotting: Analyze protein lysates from transduced HEK293T cells to determine if CAR protein is being produced.

• Intracellular Staining: Use flow cytometry for intracellular staining to detect CAR expression within the cells, which can help confirm if the CAR protein is being synthesized but not reaching the surface.

5. Assess Post-Translational Modifications

• Glycosylation: Check if the CAR requires specific post-translational modifications for proper folding and surface expression.

• Folding and Stability: Ensure that the CAR is properly folded and stable. Some CAR constructs may require chaperones or specific conditions for optimal expression.

6. Surface Marker Analysis

• Flow Cytometry: Use flow cytometry with appropriate antibodies against your CAR to assess surface expression. Ensure that your antibodies are specific and validated for use in HEK293T cells.

• Blocking Non-Specific Binding: Include appropriate controls and blocking steps to reduce non-specific binding of antibodies during flow cytometry.

7. Evaluate CAR Construct Design

• Promoter Choice: Ensure that the promoter used in your lentiviral construct is suitable for HEK293T cells.

• CAR Domain Configuration: Check if the design of your CAR (e.g., spacer regions, transmembrane domains) is optimal for expression in HEK293T cells.

8. Cell Culture Conditions

• Cell Density: Ensure that cells are at an appropriate density during transduction, as overcrowding can affect transduction efficiency.

• Culture Medium: Verify that the culture medium supports optimal cell growth and transgene expression.

9. Use of Selection Markers

• If your CAR construct includes a selection marker (like a fluorescent protein or antibiotic resistance), ensure that you are selecting adequately for successfully transduced cells.

10. Alternative Cell Lines

• If issues persist, consider testing other cell lines that might be more amenable to CAR expression, such as other human embryonic kidney cell lines or different types of adherent.

Dear Jyoti,

I totally agree with Maryam’s answers, these are good advices, and I would consider especially the first point concerning Transduction efficiency. To this end, I would like to propose you to try our LentiBlast Premium Transduction enhancer.

LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.

It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience.

LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for your experiment.

Some examples of publications with LentiBlast Premium:

• HEK293: Brown AL. Sci Rep . 2021 Oct 25;11(1):21008.
• Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
• Hematopoietic Stem Cells: Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
• HMEC: Nassour J. Nature . 2023 Feb;614(7949):767-773.
• Myeloid progenitor: Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
• T lymphocytes CD4+: Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
• THP-1: Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.

you can have more infos at: https://ozbiosciences.com/lentivirus-retrovirus/113-418-lentiblast-premium-transduction-enhancer.html#/12-capacity-500_l

Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGateor directly at: [email protected]; I will be glad to send you a sample !

best regards,

Cedric