Introduction

RNA interference (RNAi) has been used in a wide range of organisms to analyze gene function. RNAi was adapted to mammalian cells following the discovery that transfected small double-stranded RNAs can target the degradation of specific mRNAs (1). Synthetic duplex RNAs have a short half-life and therefore are not adapted for long-term studies or permanent knockdown of gene activity. Plasmid vectors that transcribe short hairpin RNAs (shRNAs) were then developed as alternative tools to suppress gene expression in mammals (2,3,4,5,6,7). These vectors usually contain the H1 or U6 promoters that transcribe a sequence encoding the sense and antisense RNA strands separated by a short loop. Transcription of this RNA terminates after synthesis of the second strand—at the TTTTT termination signal for RNA polymerase III. The synthesized RNA will fold into a stem-loop precursor, the shRNA, that is cleaved into small inhibitory RNAs (siRNAs) that enter the RNAi cellular machinery (8). The better understanding of the cellular RNAi machinery allowed substantial improvements in the efficiency of shRNA vectors. For instance, the observation of thermodynamic asymmetry in siRNAs (9,10) resulted in the development of rules predicting shRNA sequences with improved efficiencies. Recently, shRNAs that mimic a natural microRNA (miRNA) primary transcript were shown to be more efficient for suppression of gene expression (11). The shRNA technology was successfully applied to a wide series of cell lines, including embryonic stem (ES) cells (12). Understanding the biology of pluripotent ES cells is a great challenge for the future, as these cells might represent the best material for regenerative cell therapy. Loss-of-function analysis of genes using the shRNA method will provide invaluable information on their function in the maintenance of stem cell identity or their capacity to differentiate.

Here, we have set out to optimize the use of shRNA technology in ES cells by testing constructs derived from the H1 promoter. We show that a new shRNA vector, pHYPER, is 4-fold more active in ES cells than the widely used 0.2-kb H1-based shRNA plasmid. We provide evidence that pHYPER is highly efficient for the generation of stable knockdown ES cell lines and for short-term transfection experiments.

Materials and Methods

DNA Constructs

All vectors express a 23-nucleotide hairpin-type shRNA with a 6-nucleotide loop. The sequence for the green fluorescent protein (GFP) and p150CAF-1 shRNAs were described in Reference 13 and Reference 14. The construction of pHYPER and pShRNA0.2 plasmids is described in the supplementary materials (available online at www.BioTechniques.com). We used circular plasmids for transient transfection experiments, and linear, XhoI-digested plasmids for the generation of stable knockdown ES cell clones. DNA fragments were agarose gel-purified in order to remove the plasmid sequences (AmpR and Ori) prior to electroporation in ES cells.

Embryonic Stem Cells

LTP20 cells were derived from blastocysts obtained from C3H/HeJ females bred to 129Sv males, as previously described (15). LTM7 cells were described previously (14). LTP20 ES cells were transfected by electroporation with a plasmid expressing enhanced GFP (EGFP) under the control of the pCAGGS promoter (16). After a week under hygromycin selection, brightly fluorescent GFP expressing ES colonies were selected for amplification. We further selected ES clones that remained brightly fluorescent during embryoid bodies' formation and differentiation. One of these cell lines, ES-GFP1, was used in all subsequent electroporation experiments.

Epifluorescence Analysis of GFP Depletion by RNA Interference

Mouse ES cells were cultured as previously described (17). shRNA plasmid vectors were purified using a NucleoSpin® plasmid kit (Macherey-Nagel, Easton, PA, USA). ES-GFP1 cells (2×10⁷) were electroporated (250 V with a capacitance of 500 µF) with 10 µg shRNA vector, plated, and cultured for 24 h in the absence of selection. Cells were further cultured for an additional 5 days in the presence of puromycin (2 µg/mL). ES cell colonies were observed using a Leica MZFLIII epifluorescence binocular, and pictures were taken using a CoolSNAP™ camera (Princeton Instruments, Trenton, NJ, USA) and its associated software.

Northern Blot Analysis

ES cells were transfected with pHYPER and pShRNA0.2 and cultured as described in the section entitled Epifluorescence Analysis of GFP Depletion by RNA Interference. RNAs were purified by TRIZOL® reagent and chloroform extraction. We used the mirVana™ miRNA Isolation kit from Ambion (Austin, TX, USA) to further purify small RNAs. One microgram of the small RNAs' fraction was run onto a 15% acrylamide/8 M urea gel and transferred to a Hybond-N+ membrane (GE Healthcare, Piscataway, NJ, USA). Hybridization was performed at 37°C, in 6× saline-sodium phosphate-EDTA (SSPE), 0.1% sodium dodecyl sulfate (SDS), and 2× Denhart's solution, using a ³²P 5′-labeled oligonucleotide complementary to the antisense siRNA of GFP. Hybridization signals were quantified using an Instant Imager (PerkinElmer, Waltham, MA, USA).