So Many Promoters, So Little Time: Selecting Ubiquitous Promoters for Effective Gene Delivery
Keywords: Promoter, cloning, CMV, CAG, lentivirus, AAV
Promoter choice is one of the key considerations when designing a vector for gene delivery. The ideal promoter for a given vector ensures that the gene of interest (GOI) is expressed at the desired level and in the target tissues or cells (Figure 1). Dependent on the research goals, a constitutive, tissue-specific, ubiquitous, inducible, viral or non-viral promoter can be considered to drive expression of your GOI. Researchers choose ubiquitous promoters to ensure consistent gene expression across various cell types in their experiments. This article provides an overview of commonly used ubiquitous promoters and guidance on incorporating them into vector design to achieve successful gene delivery.
Figure 1. Schematic representation of promoter and other vector components
General considerations
The choice of a ubiquitous promoter for gene expression involves balancing three main factors: promoter strength, length, and desired expression level of the GOI. Promoter strength is typically guided by the biological question, tissue or cell type, and desired expression level. Strong promoters, including CMV, CAG, CBh, and EF1α, drive high levels of transcription and are used when robust protein production is desired. However, when lower, physiologically relevant expression levels are required, medium-strength promoters like PGK, SV40, MSCV, or EFS are preferred, as they provide stable, moderate expression across diverse cell types.
The length of the promoter is another important consideration and is often influenced by the cargo capacity of the vector; if the GOI is long and the vector has limited carrying capacity, a shorter (which generally tends to be weaker) promoter is used. Conversely, if the GOI is short, a longer, stronger promoter may be used. For example, in AAV systems which have a limited cargo capacity of ~ 4.7 kb, medium-strength EFS promoter (232 bp) is recommended to adjust for space, if the GOI is long. The choice of the right promoter for a specific expression system, therefore, is a delicate balance between the cargo capacity of the vector, length of the GOI, and expression level desired.
Some of the most popular ubiquitous promoters are derived from both viral and non-viral sources. CMV is one of the most widely used ubiquitous viral promoters because of its small size (589 bp) and ability to drive strong expression across many cell types. While the CMV promoter is highly effective for robust gene expression, particularly in in vitro studies, it is important to note that the CMV promoter is prone to silencing over time in certain cell types, such as stem cells. Non-viral promoters, like those derived from widely expressed genes like EF1α, provide more stable and physiologically relevant expression levels, particularly for long-term expression and in vivo applications. In addition, EF1α is less prone to silencing than CMV, making it more suitable when avoiding promoter silencing is necessary or when CMV silencing is expected. Find out more information about promoters, including strength, length, and sequence in our Vector Component Guide.
Given that the choice of promoter is unique to your specific system, application, and experimental parameters, here are some design tips summarized from our years of gene delivery experience to guide you through your choice of the right ubiquitous promoter.
Selecting promoters for viral gene delivery systems
: When using viral vectors for gene delivery, careful consideration of promoter choice is crucial to ensure successful GOI expression and virus packaging. For AAV vectors, the CMV promoter is commonly used in vitro because of its strong, constitutive expression in various cell types. However, we have observed wide silencing of the CMV promoter in AAV delivered in vivo, indicating the CMV promoter may be recognized as a viral element by the host system. Consequently, other strong promoters including CAG (1733 bp), CBh (798 bp), and EF1α (1178 bp) are preferred for AAV in vivo use for broad expression.In lentiviral vectors, the CAG promoter poses major limitations, likely due to its high GC content, which can lead to low viral titers during virus packaging. Therefore, in lentiviral vectors, promoters like CMV and EF1α are recommended instead of the CAG promoter. Another important consideration for lentiviral vector design is to avoid using the same promoter for multiple expression cassettes. Two identical promoters are prone to undergo homologous recombination for lentivirus, resulting in deletion of the gene located between them. Therefore, it is recommended to use different promoters to drive expression of multiple genes, or to express multiple genes in a polycistron using linkers like IRES or 2A, to reduce the risk of recombination.
Selecting promoters for your expression target
(GOI vs marker): When designing vectors with both transgenes and selection markers, selecting the right promoter for each ORF is crucial. For GOIs, high-level expression is often desired, so strong promoters like CMV or CBh are commonly used. Transgene expression may also require enhancers, silencers, and other regulatory elements all of which need to fit within the vector's cargo capacity, therefore smaller promoters are ideal. Selection markers are primarily used for identifying or selecting positively transfected/transduced cells, so moderate expression is typically sufficient. Since markers must be expressed in all targeted cell types and are part of a larger construct, smaller promoters are preferred to save space. Depending on the expression system, smaller, medium-strength ubiquitous promoters are ideal for markers, as they provide stable, moderate expression across a wide range of cell types.Selecting promoters when expressing toxic genes
: It is important to note that using a strong promoter to drive gene expression is not always beneficial as it can lead to cellular toxicity or other adverse physiological consequences. For example, in lentiviral vectors, the Gag and Pol genes are essential for viral particle production, but when Gag is expressed at high levels it can lead to excessive particle formation causing depletion of cellular resources, while overexpression of Pol can interfere with DNA replication and repair, leading to genomic instability. Overexpression of oncogenes like Myc can drive uncontrolled cell proliferation and trigger cellular stress pathways, resulting in cell death. Therefore, when cloning toxic genes, weaker promoters tend to be preferred; for example, we found that a lentiviral vector containing a medium-strength promoter driving mouse Foxn1 (proliferation modulator gene) produced a tenfold higher titer compared to the same vector using a strong promoter.Selecting promoters for small non-coding RNAs
: Pol III promoters, such as U6 and H1, are commonly used to produce guide RNAs (gRNA) or shRNAs in vector-based gene knockdown/knockout systems. Pol III specializes in transcribing small non-coding RNAs that are typically short and lack the 5' caps and 3' polyA tails necessary for mRNA stability, nuclear export, and translation, making Pol III a suitable promoter for small non-coding RNAs but an unsuitable promoter for expression of protein-coding genes.| Not recommended | Why it is not recommended |
|---|---|
| Using CMV promoter in AAV vectors intended for ubiquitous expression in vivo | Frequent silencing in host cells in vivo |
| Using CAG promoter in lentiviral vectors | Significantly reduced packaging efficiency and viral titer |
| Using same promoter to drive two genes in lentiviral vectors | Frequent recombination between promoters leading to deletion of sequence between promoters |
| Pol II promoter used to drive small non-coding RNA expression | Failure of transcription |
| Un-optimized toxic genes | Host cell, E. coli, or packaging cell death, low titers |
Summary
The right choice of promoter during vector design is crucial for successful gene delivery. Promoter choice depends on several factors including the vector system, transgene type, and desired expression level. Particularly for gene delivery using AAV or lentiviral systems, the right promoter can enhance efficiency, stability, and safety, ensuring experimental success without unintended side effects. Many of the considerations discussed here are incorporated in the various systems in the Vector Design Studio, and our design team is always on hand to help.
| When using | Promoter recommendation | Key considerations |
|---|---|---|
| AAV | CAG, CBh, EF1α | Avoid silencing in target cell types |
| Lentivirus | CMV, EF1α | Avoid high GC content and recombination |
| Transgene | CMV, CBh | Optimize expression and regulation |
| Marker | PGK | Size can be prioritized |
| Short non-coding RNA | U6 or H1 (Pol III) | Avoid Pol II promoters |
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