The pAd/BLOCK-iT™-DEST RNAi Gateway™ Vector can be used for the efficient delivery and transient expression of a short hairpin RNA (shRNA) in vivo from an adenoviral vector. A novel cloning process places an ∼50-bp DNA oligonucleotide immediately following a U6 pol III promoter into the BLOCK-iT™ U6 entry vector. After efficient recombination of the entry vector into the pAd/BLOCK-iT™-DEST vector, followed by viral production and transduction, the shRNA driven by the U6 promoter can be transiently expressed in most dividing or non-dividing mammalian cell types. The shRNA generated avoids the hosts defense mechanism and will be effective at producing the RNAi gene knockdown response.

The pAd/BLOCK-iT™-DEST vector (Figure 1) offers:

• A promoterless region surrounded by attR sites for efficient recombination with the attL-flanked U6 Gateway™ entry vector containing the RNAi cassette or any attL-flanked promoter and gene sequence
• All of the required components for efficient adenoviral packaging and delivery of the shRNA of interest. With this vector, transient analysis of gene knockdown in both dividing and non-dividing mammalian cell types and animal models can be achieved.