Multiple RNAi Expression Cassettes

 List of expression vectors and prices

• Definitions
• Description
• Cloning Procedure
• Alternative Cloning Procedures

The number of different plasmid vectors that can be co-transfected into the cells is often limited by the total amount of DNA that can be accommodated by transfection reagents at a time.

To overcome this problem, RNAi expression vectors from RNAi Combination Toolbox™ have been specially designed to carry multiple cloning sites, which allow you to generate multiple RNAi expression cassettes on a single vector by simple steps of DNA subcloning.

The number of expression cassettes that can be generated on a single vector is virtually unlimited, which allows you to target:

1. different regions of a gene to achieve maximum knockdown effect;
2. different isoforms of a gene;
3. different members of a gene family;
4. a set of genes in a specific signaling pathway;
5. a set of genes in different signaling pathways.

Definitions

Donor:

The expression cassette(s) that will be transferred to another vector.

Recipient vector:

The vector that will receive additional expression cassette(s).

Swapping site:

Two different, yet compatible restriction sites chosen on Donor and Recipient vectors, respectively, which will be used to subclone the expression cassette(s).

Docking site:

A common unique restriction site on both Donor and Recipient vectors, which will be used to subclone the expression cassette(s).

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Description

Initially two Swapping sites and a Docking site are chosen on both Donor and Recipient vectors. The Swapping sites are two different, yet compatible restriction sites located on Donor and Recipient vectors, respectively. The Docking sites have to be identical on both vectors (Figure 1).

Figure 1. Recommended Swapping sites and a Docking site on a representative pGBlock™ vector

On Donor vector, Swapping site and Docking site should locate on different sides of the expression cassette that is about to be moved. On Recipient vector, however, they should sit on the same side of the expression cassette(s) that additional expression cassette(s) will be received.

The expression cassette(s) DNA fragment excised from the Docor vector by restriction digestion on the selected Swapping site and Docking site is isolated and subcloned into the Recipient vector at the compatible sites. As a result, the Recipient vector will carry an additional RNAi expression cassette(s).

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Cloning Procedure

Basic steps (For example, Option I in Figure 2A):

A	B
Figure 2

1. Identify the Donor expression cassette (Target B) and the Recipient vector (Target A).
2. Select two Swapping sites (SalI and XhoI) and a Docking site (HindIII) that are going to be used.
3. Perform restriction digestion with one Swapping site and the Docking site. Choose the two on different sides of Donor expression cassette (SalI and HindIII for Target B) and the two on the same side of Recipient expression cassette (XhoI and HindIII for Target A).
4. Subclone the Donor cassette (SalI/HindIII fragment of Target B) into Recipient vector (Target A vector with XhoI/HindIII restriction digestion).
5. Verification of successful subcloning by restriction digestion on Swapping sites (SalI and XhoI) for selected clones. The size of insert should be the sum of Donor and Recipient expression cassettes (Target A and Target B).

Alternatively, SalI and XhoI can be replaced by Csp45I and ClaI to perform the same subcloning procedure (Option II in Figure 2B).

By repeating the same subcloning procedure, multiple expression cassettes can be generated on a single vector (Figure 3).

Figure 3

Notably, Swapping sites have to remain consistent once a chain subcloning procedure has begun, e.g. always use SalI and XhoI in Option I; or always use Csp45I and ClaI in Option II.

Examples of multiple RNAi expression cassettes on a single vector are given in Figure 4.

A	B
Figure 4

In theory, the number of expression cassettes that can be generated into a single vector is unlimited. However, cloning of plasmid in large size is technically challenging and requires special care when performing bacterial transformation and DNA purification procedures.

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Alternative Cloning Procedures

In addition to the basic 'one-by-one' cloning procedure as described above, users are also able to perform alternative cloning strategies to facilitate generation of multiple expression cassettes on a single vector (Figure 5).

Figure 5

By following the same cloning procedure in principle, multiple expression cassettes that are already chained can be moved altogether onto another vector at once.

Multiple RNAi expression cassettes on a single vector will multiply your possibilities in the use of RNAi technology. If you have questions about the subcloning procedure, contact us for technical support.

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