Regular single AAV-shRNA construct

The regular single AAV-shRNA vector is designed to co-express a short hairpin RNA (shRNA) for gene knockdown along with a fluorescent or luminescent reporter for easy tracking of transduction efficiency.

• Constitutive shRNA Expression: Driven by a strong Pol III promoter (e.g., U6 or H1) for efficient and continuous shRNA transcription.

• Reporter Gene Expression: Typically under a Pol II promoter (e.g., CMV or EF1a), expressing a reporter such as GFP, mCherry, or Luciferase.

• Single AAV Vector Format: Both the shRNA expression cassette and the reporter gene are packaged within one AAV genome for co-delivery to the same cell.

• Applications: Gene knockdown validation; Functional studies in vitro or in vivo; Monitoring of viral transduction efficiency

miR30-based shRNA construct

The miR-30 family is among the most abundant and well-characterized microRNAs in mammalian cells, making it an ideal backbone for designing shRNA constructs that effectively mimic endogenous miRNA processing pathways.

Unlike conventional shRNAs that are primarily expressed from Pol III promoters (such as U6 or H1), miR-30-based shRNAs can be transcribed by Pol II promoters, allowing greater flexibility in experimental design. This means shRNA expression can be driven by either ubiquitous promoters (e.g., CMV, CAG, EF1α) for broad expression or by tissue-specific promoters (e.g., hSyn for neurons, TBG for liver) for targeted gene silencing in specific tissues or cell types. Pol II-driven transcription supports longer RNA transcripts and uses the polyadenylation-dependent termination pathway, making it suitable for shRNA delivery within more complex expression cassettes, such as bicistronic vectors or gene therapy constructs.

Furthermore, miR-30-based shRNA cassettes can be inserted into either the 5′ untranslated region (5′ UTR) or the 3′ untranslated region (3′ UTR) of a transgene, enabling co-expression with a reporter or therapeutic gene. This design ensures coordinated expression of both the shRNA and the gene of interest.

AAVnerGene offers custom cloning of your shRNA sequences into our miR-30-based shRNA vectors, providing a flexible platform for stable and promoter-specific expression of shRNAs — ideal for research and therapeutic applications requiring controlled gene knockdown.

Inducible shRNA Construct

In many research and therapeutic applications, precise control over gene silencing is critical — especially when targeting essential genes or studying time-dependent biological processes.

Inducible AAV-shRNA systems offer a solution by allowing researchers to regulate shRNA expression in a controlled, reversible manner. These constructs typically employ inducible Pol II promoters (such as tetracycline- or doxycycline-responsive promoters) to drive shRNA expression, often in a miR-30 backbone for efficient processing.

Key Features:

• On/Off Control: Expression of shRNA can be turned on or off by adding or withdrawing the inducer (e.g., doxycycline).

• Reduced Off-Target Effects: Minimized basal expression helps prevent unintended gene silencing.

• Suitable for In Vivo and In Vitro Studies: Can be packaged into AAV vectors for stable delivery and long-term regulation in animal models.

• Flexible Design: Compatible with tissue-specific promoters or ubiquitous systems depending on research needs.

AAVnerGene provides custom inducible AAV-shRNA vector construction, enabling researchers to precisely control gene knockdown in both research and preclinical models.

Multi-In-One AAV-shRNA construct

AAVnerGene’s proprietary technology enables the expression of multiple shRNAs from a single AAV vector. You may provide us with either your validated shRNA sequences or the target gene information for custom design. 

For example, in our Four-in-one shRNA construct, four distinct shRNAs are cloned into a single expression vector, with different pol III promoter.  

• Knockdown of a Single Gene with Four Different shRNAs in One Vector
  Four shRNAs, each targeting a different site on the same mRNA, are cloned into a single vector — increasing the likelihood of effective gene silencing.

• Simultaneous Knockdown of Up to Four Different Genes with One Vector
  Validated shRNAs targeting 2 to 4 different genes are combined into a single plasmid, enabling the concurrent downregulation of multiple genes in one experiment.

The AAV-shRNA selection kits come with 4 shRNA, 1 control shRNA, and a GOI-mCherry fusion reporter plasmid, offering a comprehensive toolkit for effective gene knockdown and real-time monitoring of transduction and expression. shRNA can be expressed using U6 or H1 promoters, or by incorporating shRNA sequences into a miR-30 expression cassette driven by a Pol II promoter (such as CMV or EF1a). For instance, if the customer chooses the pAAVtri-CMV-EGFP plasmid as the backbone, we will place the U6/H1-shRNA cassette upstream of the CMV promoter or position the miR-30-shRNA cassette in the 5’UTR or 3’UTR. Our team will design the shRNA sequences and generate the corresponding AAV-shRNA plasmids. Additionally, we will create a GOI-mCherry fusion protein as a secondary reporter. Customers only need to provide the GOI information or sequence to be silenced. Co-transfection of the shRNA plasmid and the GOI-mCherry plasmid allows researchers to quickly assess the effectiveness of each shRNA. EGFP expression indicates transfection efficiency, while mCherry expression measures the knockdown efficiency of each shRNA.