Stop–go: Cells (green) silenced with a viral RNAi vector. Credit: INVITROGEN

Kits and reagents for making the various types of RNA interference (RNAi) constructs and getting them into cells are now widely available. The simplest options are designed to enable the negatively charged, chemically synthesized small interfering RNA (siRNA) molecules to enter cells more easily. One way is to use electroporation. Alternatively, the siRNA can be encapsulated in lipids or polymers to overcome the repulsive charges of the cell membrane, as in kits supplied by companies such as Ambion in Austin, Texas, Invitrogen in Carlsbad, California, and QIAGEN in Venlo, the Netherlands. QIAGEN has developed siRNA sets against specific functional gene families and pathways, such as those leading to apoptosis and cancer. Their Human Druggable Genome siRNA Set targets 5,000 human genes of therapeutic interest. For high-throughput siRNA-based screening, QIAGEN offers an RNAi human/mouse control kit that provides all the reagents necessary for successful start-up. Oligonucleotide producers now also make siRNAs or their DNA equivalents to order, and siRNA manufacturers such as Dharmacon in Dallas, Texas, MWG Biotech in Ebersberg, Germany, and Proligo in Boulder, Colorado, offer free online design services.

Transfected siRNAs achieve significant gene ‘knock-down’ for some 3–7 days before being naturally degraded. This is usually sufficient for studying the immediate effects of inhibiting gene expression, when screening for drug targets, for example, but is likely not to be sufficient for RNAi-based therapy.

An effect lasting for weeks can be obtained with expression systems based on plasmid or viral vectors carrying DNA versions of the interfering RNA. The DNA is transcribed and the transcript processed into siRNA within the cell. In many of these systems the constructs are expressed as short double-stranded RNA ‘hairpins’ (shRNA), which are then cleaved by cellular enzymes to produce functional siRNAs. Vectors based on adenoviruses and lentiviruses also enable RNAi to be extended to a wider range of non-dividing primary cells than when transfecting with siRNA or a plasmid. Kits for constructing adenoviral and lentiviral vector RNAi expression systems are supplied by Promega in Madison, Wisconsin, BD Biosciences in San Jose, California, Imgenex in San Diego, California, Ambion and Invitrogen.

JULIE CLAYTON