Cloning of short hairpin RNAs for gene knockdown in mammalian cells

RNA interference (RNAi) has become a methodology of choice for knocking down gene expression in a variety of biological systems(1,2). The demonstration, in mammalian in vitro systems, of gene silencing using double-stranded RNA (dsRNA) products <30 base pairs (bp) in length has placed RNAi at the forefront of gene manipulation techniques in somatic cells(3-6). Two types of dsRNA triggers are now commonly used to evoke RNAi in mammalian cells: (i) chemically or in vitro-synthesized small interfering RNAs (siRNAS)(3,4) and (ii) short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters(2). We and others have chosen to explore the shRNAs for several reasons: first, the considerable cost of chemically synthesized siRNAs; second, the possibility of enforceable and stable expression of shRNAs; and third, the availability of applications of expression constructs in primary cell types (for example, using retroviruses) and in whole organisms (for example, in mouse). We have developed a system to drive expression of shRNAs by placing them under the control of the human RNA polymerase III U6 small nuclear RNA (snRNA) promoter, which normally controls expression of small RNAs in cells. This system has now been demonstrated to be effective both in vitro(2,7-9) and in vivo transiently in mouse(10), stably during hematopoiesis(9) and stably in the generation of transgenic mice(11,12). The pSHAG-MAGIC2 (pSM2) cloning vector (Fig. 1) is roughly equivalent to pSHAG-MAGIC1 (ref. 13) with a few notable exceptions. First, the new cloning strategy is based on the use of a single oligonucleotide that contains the hairpin and common 5' and 3' ends as a PCR template (Fig. 2). That is, the oligonucleotide itself serves as template and is amplified by PCR using universal primers that contain an XhoI site (within the 5 primer) and an EcoRI site (within the 3' primer) to facilitate cloning (Fig. 3). The resulting PCR fragments are then cloned into the hairpin cloning site of the vector pSM2, the clones are verified by sequencing, and the construct is introduced into the appropriate cell lines, where expression of the miR-30-styled hairpins is driven by the human U6 promoter.