Lentiviral and adenoviral delivery of RNAi vectors

Having evolved to proficiently deliver nucleic acids to cells, viruses offer a means to reach hard-to-transfect cell types for protein overexpression or knockdown. Adenoviral, oncoretroviral, and lentiviral vectors have been used extensively for delivery in cell culture and in vivo (Table 1).

Table 1. Choose the best viral system for your experiments.

Adenoviruses are DNA viruses that can transiently transduce nearly any mammalian cell type.  The adenovirus enters target cells by binding to the coxsackie adenovirus receptor (CAR). After binding to the CAR, the adenovirus is internalized via integrin-mediated endocytosis followed by active transport to the nucleus where its DNA is expressed episomally.

Oncoretroviruses and lentiviruses are positive-strand RNA viruses that stably integrate their genomes into host cell chromosomes. When pseudotyped with an envelope that has a broad tropism, such as vesicular stomatitis virus glycoprotein (VSV-G), these viruses can enter virtually any mammalian cell type. However, the oncoretroviruses depend upon nuclear membrane breakdown during cell division to transduce cells.  In contrast, lentiviruses are more versatile tools, as they use an active nuclear import pathway to transduce nondividing cells.

For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. To accurately determine the efficacy of knockdown from an RNAi vector in a population of cells, it is critical to deliver the RNAi vector to as many cells as possible. Otherwise, when knockdown is measured by quantitative real-time PCR (qRT-PCR) or western analysis, the background of mRNA or protein in nontransfected cells will make the knockdown appear less effective than it actually is. Viral delivery can be the best option in virtually any mammalian cell type, including hard-to-transfect, primary, and even nondividing cell types. Conveniently, lentiviral delivery systems are available for both shRNA and miR RNAi vectors, and an adenoviral delivery system is available for shRNA vectors (Table 2).

The procedure for using both RNAi viral systems (Figure 1):

1. Clone the double-stranded DNA oligo encoding an shRNA or miR RNAi into one of the BLOCK-iT entry (shRNA) or expression (miR RNAi) vectors.
2. Transfer the RNAi cassette into the adenoviral (shRNA only) or lentiviral destination vector by Gateway recombination.
3. Transfect RNAi vectors into the viral producer cells to produce viral stocks, which can be used immediately or stored at –80°C
4. Harvest viral supernatants and determine the titer (amplify adenoviral stocks if desired)
5. Transduce lentiviral or adenoviral stocks to any cell type

The product manuals for the Adenoviral and Lentiviral expression systems have more detailed protocols for generating and titering Adenoviral and Lentiviral stocks.

The first step for successful transductions is to choose the system for your experimental goals (Table 1). We provide kits to clone and produce adenoviral and lentiviral stocks as well as offering services to clone these viral vectors and produce the viral stocks.  The BLOCK-iT Pol II miR RNAi Vector system and the BLOCK-iT shRNA vector system are not compatible with each other and employ different lentiviral and adenoviral vector backbones.  Once you have your miR RNAi or shRNA sequence in a lentiviral or adenoviral backbone, producing the viral stocks follows the same protocols for both vector systems.

Table 2. Choose a lentiviral or adenoviral RNAi system.