I asked chatgpt and got this. Hope it help:

1. **Verify the Lentiviral Titer**:

- Before transduction, make sure to quantify the lentiviral titer accurately. This will help you determine the actual MOI (multiplicity of infection) that you're working with.

2. **Check for Cell Viability**:

- Ensure that the Jurkat cells are healthy and viable before transduction. Healthy cells are more likely to be successfully transduced.

3. **Optimize Spinoculation Conditions**:

- The spinoculation conditions (speed, time, and temperature) can be crucial. You might want to try different spinoculation parameters to see if that improves transduction efficiency.

4. **Optimize Polybrene Concentration**:

- While you've mentioned using 8 µg/ml of Polybrene, you might want to try a range of concentrations (e.g., 4 µg/ml, 6 µg/ml, etc.) to see if there's an optimal concentration for your specific cell line.

5. **Consider a Longer Incubation Period**:

- Sometimes, extending the time after transduction before checking for expression (beyond 72 hours) can be beneficial.

6. **Check for Inhibition of Transduction**:

- Some components in the media or the serum used may inhibit transduction. Try transducing in different media or serum-free conditions to see if that makes a difference.

7. **Verify EF-1a Promoter Activity in Jurkat Cells**:

- Make sure that the EF-1a promoter is active in Jurkat cells. It's possible that this promoter might not work as efficiently in Jurkat cells compared to 293LTV cells.

8. **Consider Other Lentiviral Systems**:

- If all else fails, you might want to explore alternative lentiviral packaging systems. Different systems might work better in specific cell lines.

9. **Check for Pre-existing Immunity**:

- Jurkat cells are derived from a T-cell leukemia and might have some innate resistance or factors affecting lentiviral transduction. Ensure there are no pre-existing immune responses inhibiting transduction.

10. **Test Different Volumes of Virus**:

- Try transducing with different volumes of concentrated lentivirus. Sometimes, using a lower volume but higher titer can lead to better results.

11. **Evaluate CAR and Reporter Expression Separately**:

- Check the expression of CAR and the reporter gene separately to see if one is affecting the expression of the other.

12. **Positive Selection**:

- If possible, consider introducing a selectable marker along with your genes of interest. This could help in isolating cells that have been successfully transduced.

Remember that optimizing transduction can be a trial-and-error process, and it might take some experimentation to find the best conditions for your specific system. Also, consider consulting with colleagues or experts who have experience with lentiviral transduction in Jurkat cells. They might have specific insights or tips for your particular situation.

@Truong Nguyen

Thank you for your answer

Hi Marzieh Mazinani,

I would suggest you to try a well-known plasmid such as GFP. It would be best to do the viral packaging and transduction to Jurkat cells simultaneously with your CAR plasmid. Also, add an additional cell line (be sure it is easily transduced). So that you can differentiate where the problem is. Hope it helps!

@Didem Tecimel

Thank you for your suggestions

Likely related to your insert size. The larger your inserts, the lower your MOI. Every kb increase reduces your titer by roughly one half. You could scale up and concentrate your virus to achieve a higher MOI.