Cell culture, vectors and virus transduction
Oct4-GFP MEFs were derived from mice carrying an IRES–EGFP fusion cassette downstream of the stop codon of pou5f1 (Jackson Lab, Stock#008214) at E13.5. MEFs were cultured in DMEM (Invitrogen, 11995-065) with 10% FBS (Invitrogen) plus glutamine and NEAA. Only MEFs at passage of 0–4 were used for iPS induction. pMX-Oct4, Sox2, Klf4 and cMyc were purchased from Addgene. pMX-HA-p21 was generated by inserting N-terminally tagged-p21 into the pMX EcoRI site. pLKO-shRNA clones were purchased from Open Biosystems. To generate retrovirus, PLAT-E cells were seeded in 10 cm plates, and 9 μg of each factor was transfected the next day using Lipofectamine (Invitrogen, 18324-012) and PLUS (Invitrogen, 11514-015). Viruses were harvested and combined 2 days later. For iPS induction, MEFs were seeded in 12-well plates and transduced with ‘four factor' virus the next day with 4 μg/ml polybrene. One day later, the medium was changed to fresh MEF medium, and 3 days later it was changed to mES culture medium supplemented with LIF (Millipore, ESG1107). GFP+ colonies were picked at day 14 post-transduction, and expanded clones were cultured in DMEM with 15% FBS (Hyclone) plus LIF, thioglycerol, glutamine and NEAA. Irradiated CF1 MEFs served as feeder layers to culture mES cells and derived iPS clones. To generate shRNA lentivirus, shRNA lentiviral vectors were co-transfected into 293FT cells together with the pPACK-H1 packaging system (SBI, LV500A-1). Lentiviruses were harvested at day 2 after transfection and centrifuged at 4000 r.p.m. for 5 min at room temperature. shRNA virus was added together with four factor virus at a volume ratio of 1:1:1:1:1.
miRNA and siRNA transfection of MEFs
miRNA mimics and inhibitory siRNAs were purchased from Dharmacon. To transfect MEFs, miRNA mimics or inhibitors were diluted in Opti-MEM (Invitrogen, 11058-021) to the desired final concentration. Lipofectamine 2000 (Invitrogen, 11668-019) was added to the mix at 2 μl/well in 12-well plates, which were incubated for 20 min at room temperature. For 12-well transfections, 80 μl of the miR mixture was added to each well with 320 μl of Opti-MEM. Three hours later, 0.8 ml of the virus mixture (for iPS) or fresh medium was added to each well and the medium was changed to fresh MEF medium the next day.
Total cell lysates were prepared by incubating cells in MPER buffer (PIERCE, 78503) on ice for 20 min, and then cleared by centrifugation at 13 000 r.p.m. for 10 min. An equal volume of lysates was loaded onto 10% SDS–PAGE gels, and proteins were transferred onto PVDF membranes (Bio-Rad, 1620177) using the semi-dry system (Bio-Rad). Membranes were blocked with 5% milk in TBST for at least 1 h at room temperature or overnight at 4°C. Antibodies used include anti-p21 (BD, 556430), anti-mNanog (R&D, AF2729), anti-h/mSSEA1 (R&D, MAB2156), anti-HA (Roche, 11867423001), anti-mAgo2 (Wako, 01422023), anti-Dicer (Abcam, ab13502), anti-Drosha (Abcam, ab12286), anti-Actin (Thermo, MS1295P0), anti-AFP (Abcam, ab7751), anti-β III tubulin (R&D systems, MAB1368), anti-TGBR2 (Cell signaling, 3713s) and anti-α actinin (Sigma, A7811).
mRNA and miRNA qPCR
Total RNAs were extracted using Trizol (Invitrogen). After extraction, 1 μg total RNA was used for RT using Superscript II (Invitrogen). qPCR was performed using a Roche LightCycler480 II and the Sybr green mixture from Abgene (Ab-4166). Mouse Ago2, Dicer, Drosha, Gapdh and p21 primers are listed in Supplementary Table 2
. Other primers were previously described (Takahashi and Yamanaka, 2006
). For miRNA quantitative analysis, total RNA was extracted using the method above. After extraction, 1.5–3 μg of total RNA was used for miRNA reverse transcription using QuantiMir kit following the manufacturer's protocol (SBI, RA420A-1). RT products then were used for qPCR using the mature miRNA sequence as a forward primer together with the universal primer provided with the kit.
Cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min. Fixed cells were permeabilized with 0.1% Triton X-100 for 5 min. Cells were then blocked in 5% BSA in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Primary antibody was diluted from 1:100 to 1:400 in 2.5% BSA PBS containing 0.1% Triton X-100, according to the manufacturer's suggestion. Cells were stained with primary antibody for 1 h and then washed three times with PBS. Secondary antibody was diluted 1:400 and cells were stained for 45 min at room temperature.
EB formation and differentiation assay
iPSCs were trypsinized into a single cell suspension and the hanging drop method was used to generate EBs. For each drop, 4000 iPSCs in 20 μl EB differentiation medium were used. EBs were cultured in hanging drops for 3 days before being reseeded onto gelatin-coated plates. After reseeding, cells were further cultured until day 14 when beating areas could be identified.
Promoter methylation analysis
CpG methylation of the Nanog and Pou5f1 promoters was analysed following procedures described elsewhere (Takahashi and Yamanaka, 2006
). Briefly, genomic DNA of derived clones was extracted using a Qiagen kit. In total, 1 μg DNA was then used for genome modification analysis following the manufacturer's protocol (EZ DNA Methylation—Direct Kit, Zymo Research, D5020). After modification, PCR of selected regions was performed, and the products were cloned into pCR2.1-TOPO (Invitrogen). Ten clones were sequenced for each gene.
Teratoma formation and chimera generation
To generate teratomas, iPSCs were trypsinized and resuspended at a concentration of 1 × 107 cells/ml. Athymus nude mice were first anesthetized with Avertin, and then ~150 μl of the cell suspension was injected into each mouse. Mice were checked for tumours every week for 3–4 weeks. Tumours were harvested and fixed in zinc formalin solution for 24 h at room temperature before paraffin embedding and H&E staining. To test the capacity of derived iPSC clones to contribute to chimeras, iPSCs were injected into C57BL/6J-Tyr(C−2J)/J (albino) blastocysts. Generally, each blastocyst received 12–18 iPSCs. ICR recipient females were used for embryo transfer. The donor iPSC cells are in either agouti or black colour.
mRNA microarray analysis
miR-93 and siControl were transfected into MEFs and total RNAs were harvested at 48 h post-transfection. mRNA microarray was carried out by Microarray facility in Sanford-Burnham Institute. Gene lists for both potential functional targets (fold change >2, P<0.05) and total targets (fold change >25%, P<0.05) were generated by filtering through volcano maps. Gene lists were then used for ontology analysis using GeneGo software following guidelines from the company.
Dual luciferase assay
3′UTR of both p21 and Tgfbr2 were cloned into XbaI site of pGL3 control vectors. For each well of 12-well plates, 200 ng of resulted vectors and 50 ng of pRL-TK (renilla luciferase) were transfected into 1 × 105 Hela cells which were seeded 1 day before the transfection. In total, 50 nM of miRNAs were used for each treatment and cell lysates were harvested at day 2 post-transfection. In total, 20 μl of lysates were then used for dual luciferase assay following the manufacturer's protocol (Dual-Luciferase® Reporter Assay System Promega, E1910).
Cell proliferation assay
In total, 3000 MEFs were seeded in each well in 96-well plates and transduced with 4F virus and shRNA lentivirus (or transfected with miRNA inhibitors). Starting from day 1 post-transduction/transfection, every 2 days, cells were incubated with mES medium containing Celltiter 96 Aqueous one solution (Promega, G3580) for 1 h in tissue culture incubator. Absorbance at 490 nm was then measured for each well using plate reader and collected data were used to generate relative proliferation curve using signal from day 1 post-transduction/transfection as the reference.