The pLenti6/BLOCK-iT™-DEST expression vector provided in the BLOCK-iT™ Lentiviral RNAi Expression System can be used to efficiently introduce and stably express short hairpin RNA (shRNA) in vivo from a lentiviral 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. The oligonucleotide is designed to express RNA that forms a stem-loop structure containing the sense and antisense regions of your target gene of interest. This shRNA is then recombined into the pLenti6/BLOCK-iT™-DEST vector. After viral production and transduction, the shRNA driven by the U6 promoter becomes stably integrated as an RNAi cassette. The shRNA generated avoids the hosts defense mechanism and will be effective at producing the RNAi gene knockdown response (Figure 1).

The pLenti6/BLOCK-iT™-DEST vector (Figure 2) offers:

• attR sites for efficient recombination with the attL-flanked U6 Gateway™ entry vector containing the RNAi cassette
• All of the required components for efficient lentiviral packaging and delivery of the shRNA of interest
• Blasticidin selection marker for fast, efficient selection of stable cell lines expressing the shRNA Using the BLOCK-iT Lentiviral RNAi Expression System, long-term analysis of gene blocking in both dividing and non-dividing mammalian cell types and animal models can be achieved.

The BLOCK-iT™ RNAi U6 Entry Vector Kit allows streamlined cloning of shRNA target sequences for testing in transient experiments. Selected RNAi expression cassettes are quickly and efficiently recombined from the BLOCK-iT™ RNAi U6 entry vector into the pLenti6/BLOCK-iT™-DEST vector via a standard Gateway™ LR recombination reaction (Figure 3).