Hi Jaime,

As no one answered, I can provide you with some references you might like to look at even though I am not totally qualified for that:

Optimized gene silencing by co-expression of multiple shRNAs in a single vector http://www.ncbi.nlm.nih.gov/pubmed/20217547

A comparison of multiple shRNA expression methods for combinatorial RNAi http://www.gvt-journal.com/content/9/1/9

Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene http://www.ncbi.nlm.nih.gov/pubmed/18367095

Hope this helps until you receive better feedback. Good luck!

Hi Norbert,

Thanks for the references. I guess the idea is to subclone each of the shRNA+its own promoter in the same vector and you get it. Thanks again, that should work! Jaime

Hi,

I am using psiRNA DUO plasmid from Invivogen.

http://www.invivogen.com/psirna-duo

Hey Martin,

That one sounds good too, thanks!

Hey everybody,

in my experience there wasn't any problem to express 4 shRNAs from one plasmid with the H1-Promotor. Probably even more will work.

In fact we have just published a vector system especially for that purpose: Article A simple approach for multi-targeted shRNA-mediated inducibl...

Feel free to contact me if you need more information or the vectors.

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and deeply engrossed in the advancement of your research, particularly in the realm of RNA interference (RNAi) technologies. Your inquiry regarding the use of a single vector to express two short hairpin RNAs (shRNAs) is both timely and indicative of the innovative approaches sought in the field to enhance gene silencing efficiency and specificity. Below, I provide an overview of the concept, utility, and considerations associated with employing a single vector for dual shRNA expression.

Concept of Dual shRNA Expression

The idea of co-expressing two shRNAs from a single vector is grounded in the objective to simultaneously target multiple genes or different regions of the same gene, thereby potentially increasing the effectiveness of gene silencing, overcoming issues of viral load limitations, and reducing the likelihood of escape mutations.

Design and Construction

Applications and Advantages

• Synergistic Silencing: Targeting multiple genes or multiple sites within a gene can lead to synergistic effects, enhancing the overall silencing efficacy.
• Reduced Viral Load: Co-expression from a single vector minimizes the number of viral particles required for effective transduction, important for in vivo applications where viral load is a concern.
• Versatility: This approach offers flexibility in targeting complex diseases or pathways involving multiple genes.

Considerations and Best Practices

• shRNA Design: Careful design of each shRNA to ensure specificity and minimize off-target effects is paramount. Bioinformatics tools can aid in shRNA sequence selection.
• Vector Size Constraints: The size of the insert can affect viral packaging efficiency. It is essential to keep the overall vector size within the optimal range for the chosen viral system.
• Experimental Validation: Rigorous in vitro and in vivo validation of the dual shRNA expression system is necessary to confirm the efficiency of gene silencing and to assess any unforeseen effects on cell physiology.
• Regulatory Elements: Incorporating appropriate regulatory elements, such as insulators, can help maintain consistent expression levels and prevent transcriptional interference between the two shRNAs.

Conclusion

Utilizing a single vector for the expression of two shRNAs represents a promising strategy for enhancing the scope and efficiency of gene silencing applications in research and therapeutic contexts. By meticulously designing the vector and shRNAs and conducting thorough validation, researchers can leverage the advantages of this approach to address complex biological questions.

Should you require further insights or wish to discuss the practical aspects of constructing and employing dual shRNA vectors, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the advancement of your research projects.

Warm regards.

Check out this protocol list; it might provide additional insights for resolving the issue.