Knock-Down of Gene Expression in Hematopoietic Cells

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that results either in degradation of homologues mRNAs or inhibition of mRNA translation. The effector molecules which activate the RNAi pathway are small regulatory RNAs including small interfering RNAs (siRNAs) which are processed from longer dsRNAs by the RNAse III enzyme Dicer, and microRNAs (miRNAs) generated in a regulated multistep process from endogenous primary transcripts (pri-miRNA). Since, in principle, any gene can be silenced, RNAi provides a powerful tool to investigate gene function, and it is therefore a widely used gene silencing method in functional genomics. This chapter provides a collection of protocols for specific gene knock-down in hematopoietic cells by the application of short-hairpin RNAs (shRNAs) transcribed by RNA polymerase III (pol III) promoters or artificial-miRNAs (art-miRNAs) expressed from RNA pol II promoters using lentiviral vectors, respectively.