Hi Laurent: A collaborator of mine has used the vav proto-oncogene promoter for weak yet stable expression.

http://www.ncbi.nlm.nih.gov/pubmed/17534266

The ones you have mentioned are regularly used by our laboratory for various transgene expression- really depends on what your target cells are- I have used the EF1alpha. You can also try using the UCOE for the long-term benefit of avoiding silencing.

http://www.ncbi.nlm.nih.gov/pubmed/22696657

Hope this helps-

Sayandip.

Hi Laurent, in case you are interested in co-expression studies, the paper below might be relevant for you. In it. we looked into some coexpression and promoter effects a while back.

http://www.ncbi.nlm.nih.gov/pubmed/19847204

HTH

In most of cells the order of promoter strength is like CMV > or = EF1a > PGK > or = UBC. You can also use attenuated IRES. The gene expressed from IRES is about 7-10 fold lower than the same gene expressed before the IRES (directly from the promoter). Removing intron (if there is one) and Kozak sequence may help.

Thank you guys it helps a lot

Dear Colleague,

I hope this message finds you well. Your inquiry regarding the use of lentiviral vectors for transgene expression under the control of a weak promoter is a topic of considerable interest in the field of gene therapy and molecular biology research. Drawing from collective experience and existing literature, I am pleased to share insights that might assist you in navigating the challenges and opportunities associated with this approach.

Background

Lentiviral vectors are widely used for stable gene delivery due to their ability to integrate into the host genome, enabling long-term expression of the transgene. The choice of promoter is critical in regulating the level of transgene expression. While strong promoters like CMV are commonly used for high expression levels, there are scenarios where a weak promoter might be preferable to achieve low, physiological levels of expression or to minimize cytotoxicity.

Considerations for Using Weak Promoters

Challenges and Solutions

• Low Transgene Expression: If the expression level achieved is too low for practical applications, consider using a slightly stronger promoter or incorporating multiple copies of the promoter. Alternatively, vector modifications that enhance mRNA stability or translation efficiency could be beneficial.
• Cell-to-Cell Variability: Heterogeneity in transgene expression can be a concern. Sorting transduced cells based on reporter gene expression or employing single-cell techniques may help in isolating cells with desirable expression levels.
• Long-Term Expression Stability: Monitor the stability of transgene expression over time, as promoter silencing can occur, especially in dividing cells. Epigenetic modifications or the use of insulator sequences may mitigate this issue.

Conclusion

Employing lentiviral vectors with weak promoters for controlled transgene expression is a nuanced approach that requires careful planning and optimization. By considering the aspects mentioned above and proceeding with thorough experimental validation, you can effectively leverage this strategy for your research objectives.

Should you have further questions or require detailed discussion on specific aspects of your project, feel free to reach out.

Best regards,

With this protocol list, we might find more ways to solve this problem.