With pEGFP-C1 (Clontech, Palo Alto, Calif.) as the template, a 547-bp fragment encoding a portion of the EGFP gene beginning from the ATG start codon was amplified by PCR with the primers 5′-GCC GTC GAC GGT ACC TCT AGA ACG CGT GCC ATG GTG AGC AAG GGC GAG GAG-3′ and 5′-GCC GCG GCC GCG GCC CTA TTA GCC CTC GAG TAC ATG GTC GGC GAG CTG CAC GCT-3′. A set of restriction sites, SalI, XbaI, and MluI at the 5′ end and EcoRI and NotI at the 3′ end, were incorporated in each primer. After PCR amplification, the 547-bp fragment was digested with NotI and self-ligated. The dimer of the 547-bp fragment was isolated from the agarose gel, purified, ligated to the pGEMT Easy vector (Promega, Madison, Wis.), and used to transform Escherichia coli DH5α (Gibco-BRL, Rockville, Md.) competent cells. The resulting colonies were screened for inverted repeats by restriction enzyme analysis. The sequence of the plasmid harboring an inverted repeat (pGEMT-dsEGFP) was confirmed by DNA sequencing.
To generate control dsRNA, a 629-bp fragment encoding a portion of the lacZ
gene (nucleotides 1331 to 1960 from the AUG start codon) was amplified by PCR and ligated to the pGEMT Easy vector. The colonies were screened for plasmids containing the insert in the sense and antisense orientations. The plasmid pSC6-T7-Neo, encoding the T7 RNA polymerase gene under the control of the cytomegalovirus (CMV) promoter was a generous gift from M. Billeter (25
). The pActin-lacZ and pIZ/US9-GFP plasmids, encoding the lacZ
and EGFP genes under the control of the Drosophila
promoters for actin and OpIE2, respectively, were generous gifts from Gregory Hannon. The pCMV-lacZ plasmid was purchased from Clontech.
In vitro transcription of dsRNA.
The pGEMT-dsEGFP construct with an inverted repeat containing a portion of the EGFP gene was linearized with PstI at a unique site located at the 3′ end of the inverted repeat. Using the RiboMax large-scale RNA production system-T7 (Promega, Madison, Wis.), the transcription reaction was performed at 37°C for 3 h, according to the manufacturer's specifications.
Radiolabeled dsRNA was generated by incorporation of [α-32P]UTP during in vitro transcription. After performing the in vitro transcription reaction, RNase-free DNase (Promega, Madison, Wis.) was added to the reaction mixture at 1 U/μg of the template DNA and incubated for 15 min at 37°C. The transcript was further purified by extraction in phenol-chloroform-isoamyl alcohol (25:24:1) and ethanol precipitation. The pellet was washed with 70% ethanol, dried at room temperature, and resuspended in TE buffer (10 mM Tris [pH 7.5] and 1 mM EDTA). To determine the folded structure of the dsRNA, an aliquot of the RNA sample was digested using a mixture of RNase A and T1 (Ambion, Austin, Tex.) at 37°C for 30 min and analyzed on 5% nondenaturing and denaturing polyacrylamide gels containing 40% formamide and 7 M urea. For dsRNA of the lacZ gene, plasmid containing either the sense or antisense lacZ fragment was linearized by restriction enzyme SalI, located downstream of the multiple cloning site. The sense and antisense RNAs were generated separately by in vitro transcription and annealed to generate a 740-bp dsRNA fragment.
Cell culture. Drosophila
S2 cells (generous gift from G. J. Hannon) were maintained at 27°C in 90% Schneider's insect medium (Gibco-BRL, Rockville, Md.) and 10% heat-inactivated fetal bovine serum (FBS). Cells were split every 2 to 3 days to maintain exponential growth. BsrT7/5 (4
), a derivative of BHK-21 cells that express the T7 RNA polymerase (generous gift from M. Schnell), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and penicillin-streptomycin. CHO-K1 cells were maintained in F-12 medium with 10% heat-inactivated FBS. Mouse embryonic stem (ES) cells AB2.2 (Stratagene, La Jolla, Calif.) were maintained in DMEM supplemented with 1,250 U of leukemia inhibitory factor (LIF) (Chemicon, Temecula, Calif.) per ml 15% FBS, 2 mM glutamine, 100 mM β-mercaptoethanol, and 1× nonessential amino acids. Mouse embryonic fibroblast STO cells (American Type Culture Collection, Rockville, Md.) were grown in DMEM with 10% FBS. The STO feeder cells were plated on dishes coated with 0.1% (wt/vol) gelatin, treated with mitomycin C (Sigma, St. Louis, Mo.) at a concentration of 10 μg/ml for 2.5 h at 37°C, and washed three times with phosphate-buffered saline (PBS). ES AB2.2 cells were plated onto mitomycin C-treated STO feeder cells and passaged every 2 days with a daily change of culture medium. For all experiments, ES cells were kept between 17 and 19 passages, counted from the time of isolation of ES cells from the inner cell mass of the blastocysts.
The day before transfection, S2 cells were plated in a 12-well plate (106
cells per well). Various amounts of the Pst
I-linearized pGEMT-dsEGFP plasmid or dsRNA generated by in vitro transcription were combined with 1 μg of the target plasmid encoding EGFP (pEGFP-C1) and 1 μg of the plasmid encoding the T7 RNA polymerase (pSC6-T7-Neo). In all transfection experiments, a constant amount of total DNA, 5 μg, was maintained by addition of the unrelated pUC19 plasmid. DNA was transfected to S2 cells by the calcium phosphate method. The plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. CHO-K1 and STO feeder cells were plated in a 12-well plate (105
cells per well) the day before transfection. Various amounts of the Pst
I-linearized pGEMT-dsEGFP plasmid or in vitro-transcribed dsRNA was combined with 1 μg of the target plasmid encoding EGFP (pEGFP-C1) and 1 μg of the plasmid encoding the T7 RNA polymerase (pSC6-T7-Neo). The DNA mixture was transfected to cells by addition of 7.5 μg of Lipofectamine (Gibco-BRL, Rockville, Md.). The same transfection protocol was used for BsrT7/5 cells except the pSC6-T7-Neo plasmid was not added, as BsrT7/5 already expresses the T7 RNA polymerase (4
ES cells were grown on feeder STO cells, trypsinized for 5 min, and pipetted extensively to prevent clumping of cells. After addition of 5 volumes of ES medium, cells were put back in the incubator for 45 min. The majority of the STO cells adhere to the plate during this incubation, and ES cells were harvested from the suspension. Various amounts of the PstI-linearized pGEMT-dsEGFP plasmid or dsRNA were combined with 1 μg of the target plasmid encoding EGFP (pEGFP-C1) and 1 μg of the plasmid encoding the T7 RNA polymerase (pSC6-T7-Neo). In all transfection experiments, a constant amount of total DNA, 5 μg, was maintained by addition of the unrelated pUC19 plasmid. A 150-μg M9 peptide (generous gift from Scott Diamond) was then added to the DNA solution in 100 μl of OptiMEM, and the DNA-M9 mixture was further incubated for 15 min at room temperature. Lipofectamine (7.5 μg) was diluted in 100 μl of OptiMEM and added to the DNA-M9 mixture for 45 min. The DNA-M9-Lipofectamine mixture was added to 3 × 105 ES cells in suspension and plated in a 12-well gelatin-coated plate containing 3 × 105 STO feeder cells pretreated with mitomycin C (Sigma, St. Louis, Mo.). The same procedure was used for transfection of ES cells without feeders except that ES cells were plated directly on gelatin plates without STO feeder cells, using medium without LIF .
Quantitation of EGFP and β-galactosidase activities.
All adherent cells were harvested 72 h after transfection by washing with PBS and scraping cells into 100 μl of ice-cold lysate buffer (91.5 mM K2HPO4, 85 mM KH2PO4, and 1 mM dithiothreitol [DTT]). The harvested cells were then subjected to a dry-ice/ethanol freezing and thawing at 37°C for three cycles and centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was stored at −70°C until use. For ES cells with feeders, 72 h after transfection, cells were trypsinized for 5 min and pipetted extensively. After addition of 5 volumes of ES medium, cells were put back in the incubator for 45 min to allow the STO cells to attach to the plate. After this procedure, ES cells constituted more than 95% of the cells in suspension. ES cells were transferred to a tube, centrifuged, and processed the same way as other cells. The protein concentration of cell lysates was measured with the Pierce reagent (Pierce, Rockford, Ill.) in a 96-well plate. For each lysate, the same amount of protein was used for the fluorescence and chemiluminescence measurements. Fluorescence was measured in relative light units (RLUs) using a 96-well black flat-bottomed plate (Corning Costar, Cambridge, Mass.) and an FL 600 microplate reader (Bio-Tek Instrument, Winooski, Vt.) with KC4 data reduction software on an external personal computer, which controls the reader function and data capture. Excitation was at 485 nm with a 20-nm band-pass filter, and emission was at 530 nm with a 25-nm band-pass filter. To account for the background, each fluorescence reading was subtracted from that of the untransfected cell lysate. The fluorescence reading of each lysate was normalized to that of the lysate prepared from cells transfected without dsRNA, either pGEMT-dsEGFP plasmid or in vitro-transcribed dsRNA.
β-Galactosidase activity was measured by histochemical staining and chemiluminescence. Cells were fixed with 1% glutaraldehyde and stained with X-Gal staining solution [0.1 M sodium phosphate (pH 8.0), 1.3 mM MgCl2, 3 mM K4Fe(CN)6, 3 mM K3Fe(CN)6, and 0.4 mg of X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) in N,N-dimethyl formamide] for 4 h. For chemiluminescence measurements, cell lysates were prepared using the Luminescent β-Galactosidase Genetic Reporter System II kit (Clontech, Palo Alto, Calif.). Lysates were analyzed in triplicate by chemiluminescence using the Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany). To account for the background, the chemiluminescence reading was subtracted from that of the untransfected cell lysate. The RLUs of each lysate were normalized to that of the lysate prepared from cells transfected without dsRNA, either pGEMT-dsEGFP plasmid or in vitro-transcribed dsRNA.
Fluorescence microscopy of S2 and ES cells.
For fluorescence microscopy, S2 cells (2 × 106 S2 cells/well of a six-well plate) were plated and transfected with 2.5 μg of pIZ/US9-GFP and an increasing amount, 0, 1.5, or 3.0 μg, of the in vitro-transcribed dsRNA by the calcium phosphate method. ES cells (6 × 105 cells/well of a six-well plate) were mixed with 2.5 μg of pEGFP-C1 plasmid and increasing amounts, 0, 1, and 2 μg, of the in vitro-transcribed dsRNA and plated on the STO feeder cells as described above. Fluorescence micrographs were taken 72 h after transfection.
Analysis of RNA by Northern blotting in ES cells.
ES cells were transfected by three plasmids as described above. Total RNA was purified by the RNAeasy Mini kit (Qiagen, Valencia, Calif.) and quantitated by UV absorbance at 260 nm. A total of 25 μg of RNA was loaded into each lane in a 0.8% formaldehyde denaturing agarose gel, and the Northern blotting was performed using the NorthernMax kit (Ambion, Austin, Tex.). The EGFP probe was a 0.7-kb fragment generated by NheI and BglII restriction enzyme digestions of plasmid pEGFP-C1, and the lacZ probe was a 2.5 kb fragment prepared by PvuII digestion of the pCMV-lacZ plasmid. The probes were labeled by [α-32P]dCTP using the Megaprime DNA labeling system (Amersham, Piscataway, N.J.). A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.
Generation of ES cells with integrated EGFP gene, transfection with dsRNA, and FACS analysis.
To produce ES cells with an integrated EGFP transgene, 2.5 × 106 AB2.2 cells were transfected with 6 μg of linearized pcDNA3-EGFP by M9 lipofection. G418 (275 μg/ml) selection was started 24 h after transfection. After 10 days of selection with G418, the surviving ES colonies were examined by fluorescence microscopy. Fluorescent colonies were picked and expanded according to established techniques. The number of integrated copies of pcDNA3-EGFP was determined by Southern blot analysis using the EGFP coding region as a probe. Several ES clones with a single copy of the EGFP gene were chosen for use in this study. ES cells were seeded at 105 cells/well of six-well plate in the presence and absence of the feeder layer and transfected with 3 μg of in vitro-transcribed dsRNA-EGFP or dsRNA-lacZ using 1.5 μg of Lipofectamine 2000 (Gibco-BRL, Rockville, Md.). The transfected cells were maintained with daily changes of medium and harvested at various time points to measure GFP fluorescence. Cells were trypsinized, centrifuged, suspended in chilled PBS, and subjected to fluorescence-activated cell sorting (FACS) analysis using a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.). Instrument settings were adjusted to separate live from dead cells, and fluorescence intensity data for 20,000 live cells were collected for each experimental time point. The relative levels of fluorescence for different samples were compared using the geometric means. Instrument settings were kept constant for all samples within each experiment. Data were analyzed using Cell Quest Software (Becton Dickinson).
Generation of small RNA fragments from dsRNA.
Cytoplasmic extracts were isolated as described previously (8
). Extracts were prepared from cells in the log phase of growth, and cytoplasmic proteins were extracted in a buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2
, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride (PMSF; Sigma, Saint Louis, Mo.), and 0.5 mM DTT (Sigma, Saint Louis, Mo.). The final dialysis was performed for 12 h in an excess volume of dialysis buffer (20 mM HEPES [pH 7.9], 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT). The protein concentration was measured by the Bradford assay. Cytoplasmic extracts (10 to 50 μg) were incubated with 30 nmol of radiolabeled dsRNA for 1 h at 30°C for S2 cells or 37°C for mammalian cells. The standard reaction was carried out in a 20-μl reaction buffer containing 20 mM HEPES, 2 mM magnesium acetate, 2 mM DTT, 1 mM ATP, 40 mM creatine phosphate, and 100 μg of creatine phosphokinase and 1 U of RNasin (Ambion, Austin, Tex.) per ml. After the reaction, samples were treated with proteinase K (1 mg/ml)–0.5% sodium dodecyl sulfate (SDS) and purified by phenol-chloroform extraction. The size of the dsRNA was examined by a 12% denaturing acrylamide gel. After completion of electrophoresis, the gel was stained with ethidium bromide. The gel was then fixed in a 30% methanol–7% acetic acid solution, dried, and exposed to X-ray film at −80°C.