Lentiviral strategies for RNAi gene knockdown

Introduction

Among the 30-40 thousand protein-encoding genes in the human genome, the function of only a fraction are known. A new tool for systematically deciphering the functions and interactions of these thousands of genes has been developed: short hairpin RNAs. These hairpin RNAs are precursors to the short interfering RNAs (siRNAs) that are the powerful mediators of RNA interference (RNAi) (1, 2). In RNAi, genes homologous in sequence to the siRNA are silenced at the post-transcriptional state.

The original reports on the design and use of short hairpin RNAs revealed that there are a variety of different hairpin structures that may give rise to effective siRNAs (3-9). These reports addressed two major limitations of short interfering RNA (siRNA) technology: the efficiency of transfection and the longevity of the silenced state. These original reports used polymerase III (pol III) promoters to transcribe the siRNAs or RNA hairpins, and later reports have shown efficacy of siRNA generation from pol II promoters (10).

Lentiviruses, such as the human immunodeficiency virus (HIV) are capable of infecting non-dividing cells, including differentiated neurons of the brain. Short hairpin RNAs can be expressed from lentiviruses, allowing for high efficiency transfection of a variety of cell types. Approaches toward lentiviral hairpin-mediated RNAi are presented, and methodologies for deriving RNAi-transgenic cells and animals are discussed.

Choice of lentiviral vector

There are a number of lentiviral vectors available for performing short hairpin-directed RNAi in mice (11-14). Some vectors encode green fluorescent protein (GFP) as a marker for infectivity and to track the RNAi knockdown cells in real time. Other vectors are available which contain antibiotic selection markers, such as puromycin, which are useful when selection is imposed in an experiment. In addition, there are conditional versions of lentiviral RNAi vectors available, where the user may opt to "turn on" or "turn off" RNAi during the course of experimentation (11, 15). Although most RNAi lentiviral vectors are freely available from academic sources, several commercial sources also provide vectors.

Design of the short RNA hairpin

One of the first steps towards using the lentiviral RNAi method is to design an effective RNA hairpin construct. Although not every hairpin construct will produce an effective RNAi response, rules have been developed that enrich for successful constructs. These rules are based on the examination of large numbers of effective constructs and thermodynamic analysis of microRNAs and effective siRNAs(16-18). Prediction algorithms for designing hairpin RNAs (and siRNAs) are available on the web (see designing RNA ), where investigators can input their gene sequence into an online-window, and a program will output the DNA oligos that need to be synthesized to make the constructs. Several institutions have undertaken large-scale efforts to create lentiviral RNAi libraries, ie. a vector for every human and mouse gene. As these programs mature, individual lentiviral RNAi constructs and even entire lentiviral RNAi libraries will be available.