All primer sequences are listed in Supplementary Table 2
. All PCR-amplified fragments were verified by sequencing.
< 2A peptide-linked 4- or 5-factor cassettes>
Mouse Oct4, Sox2, Klf4 and cMyc were PCR-amplified with primer pairs Oct4-B-U1 and Oct4-F2A-S, Sox2-X and Sox2-F2A-S, Klf4-X and Klf4-F2A-S, and Myc-X and Myc-S, respectively. Oct4 PCR product was digested with BamHI and SalI, and cloned into pBluescriptIIKS+ (pBS), resulting in pPB-Oct4F2A. Sox2, Klf4 and cMyc PCR products were cloned into a TA cloning vector (Promega), resulting in pGEMT-Sox2F2A, Klf4F2A, cMyc, respectively. To combine 4 factors in a single vector, a XhoI-SalI fragment of Sox2-F2A was first inserted into the SalI site of pBS-Oct4F2A, resulting in pBS-OfSf. Then a XhoI-SalI frament of Klf4-F2A was inserted into the SalI site of pBS-OfSf, resulting in pBS-OfSfKf. Finally, a XhoI-SalI fragment of cMyc was inserted into the SalI site of pBS-OfSfKf, resulting in pBS-4F.
To change F2A between Oct4 and Sox2 to T2A, the NsiI-AatII fragment of pBS-4F was replaced with a PCR fragment generated with a primer pair of OctT2ASox and OtS-seqL, resulting in pBS-4FT.
To further change F2A between Sox2 and Klf4 to T2A, the AatII-MscI fragment of pBS-4FT was replaced with a PCR fragment generated by fusion-PCR. Briefly, two PCRs were performed using primer pairs StK-U1 and StK-L2, and StK-U2 and StK-L1 in separate tubes. These PCR products were then column-purified and used as templates for a 2nd PCR with a primer pair StK-U1 and StK-L1. The resulting vector was called pBS-4FTT.
To add Lin28 to the 2A peptide-liked 4-factor vector, Lin28 cDNA was PCR-amplified by using Lin28-U1 and Lin28-L1 and cloned into the BamHI-EcoRI site of pBS. A T2A-Lin28 fragment flanked by BstBI and XhoI sites was amplified by PCR (primer pair MtL-U1/MtL-L1) and inserted into the BstBI-XhoI site of pBS-4FTT, resulting in pBS-4FTTL.
OSKM, OSKM*, and OSKML in correspond to 4FTT, 4FT, and 4FTTL, respectively.
<piggyBac transposon-based expression vector>
First, a CAG promoter-driven expression vector, pCAG.EBNXN, was generated by replacing the Eco
I fragment of pCAG-IP 33
with a linker containing multicloning sites (Eco
I). Second, a piggyBac
vector carrying a CAG promoter-driven expression cassette, pPB-CAG.EBNXN, was generated by inserting the Sal
HI fragment of pCAG.EBNXN into the Xho
HI site of pPB-LR.
<4-factor or 5-factor expression vectors>
Four- or five-factor expression vectors were constructed by inserting the BamHI-SalI fragment of the 4-factor construct or the BamHI-XhoI fragment of the 5-factor construct into the BglII-XhoI site of pPB-CAG.EBNXN, resulting in pPB-CAG.4F, pPB-CAG.4FT, pPB-CAG.4FTT, and pPB-CAG.4FTTL, respectively. Finally, a negative selection marker, PGK-puΔtk cassette, was excised from pFlexible with XhoI digestion and inserted into the SalI site of pPB-CAG-based factor expression vectors.
< piggyBac transposon vector with an eGFP cassette>
First, a piggyBac
transposon vector carrying the human ubiquitin C (UbC) promoter-driven expression cassette was constructed. UbC promoter and bovine growth hormone polyadenylation signal sequence (bpA) were PCR amplified with a primer pair of UbC-U and -L and bpA-U and -L, respectively. An Nhe
RI fragment of UbC promoter and an Eco
I fragment of bpA were ligated into the Nhe
I site of pPB-LR5, resulting in pPB-UbC. Second, an eGFP fragment was excised from pCX-GFP and inserted into the Eco
RI site of pPB-UbC, resulting in pPB-UbC.eGFP. Finally, PGKneo cassette was excised from PL452 34
I digestion and inserted into the Sal
I site of pPB-UbC.eGFP, resulting in pPB-UbC.eGFP-neo
MEFs were prepared from e14.5 wildtype embryos (inbred C57Bl/6-Tyr c-Brd/c-Brd for the piggyBac study, B6129S6F1 and ICRB6F1 for the retroviral study) and cultured in DMEM containing 10 % FBS (HyClone), 2 mM L-glutamine, 1 × non-essential amino acid, and 0.1 mM 2-mercaptoethanol. A germline-competent mouse ESC line (KY1.1, B6129S6F1 background; unpublished) and mouse iPSC lines were cultured on mitomycin C-treated MEFs in serum-based ESC medium (F15L), which is DMEM containing 15 % FBS (HyClone), 2 mM L-glutamine, 1 × non-essential amino acid, 0.1 mM 2-mercaptoethanol and 1000 U ml−1 LIF (Chemicon).
Reprogramming of MEFs using transposon vectors
MEFs were plated onto 6-well plates (5 × 105
cells/well) one day before transfection. The next day (day 0), 2.0 μg of pCMV-mPBase 35
and various amounts of plasmids harboring the piggyBac
transposon were transfected using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. On day 1, transfected MEFs were trypsinised and replated onto feeder layers at a split ratio of 1:18 in MEF medium. On day 2, F15L medium was applied. VPA was added to culture medium at 2 mM 19
from day 2 to day 7. The medium was refreshed every other day. On day 7, medium was changed to serum-free ESC medium (K15L), which contains 15% Knockout serum replacement (Invitrogen) instead of FBS. Medium was refreshed every other day. On day 14, colonies were either stained using the Alkaline phosphatase detection kit (Chemicon) and counted, or picked and further expanded.
Reprogramming of MEFs by retroviral vectors
Retroviral vectors (pMXs-Oct3/4, pMXs-Sox2, pMXs-Klf4, pMXs-c-Myc) 2
were obtained from Addgene. Preparation of retroviruses and infection of MEFs was described in ref. 2
. One day post infection cells were replated onto 6-well plates containing a feeder layer at 3,000 cells/well. Subsequent culture conditions are described in Supplementary Figure 2
Transposon removal from primary iPSCs
transposase-expression vector was electroporated into 2 × 106
iPSCs. Cells were maintained for 3 days to allow turnover of the puΔtk protein. 5 × 105
cells were then seeded onto 10 cm dishes containing feeder cells. The next day FIAU was added to the culture medium (0.2 μM final concentration) and selection was continued for 5 days. After an additional 5 days of culture without FIAU, the resulting colonies were picked and expanded. Transposon removal was examined by PCR with primers listed in Supplementary Table 2
and confirmed by Southern blot analysis using the 5′ terminal repeat of the piggyBac
transposon as a probe. The probe for the 5′ terminal repeat of piggyBac
transposon specifically detects the 5′ repeat and does not detect the 3′ terminal repeat in the Southern blot analysis.
GFP marking of an integration-free iPSC line
A cured iPSC line iPS25Δ1 was electroporated with 25 μg of a linearized Nanog-GFP targeting vector (unpublished, K.Y. and J.T.) in Hepes-buffered saline (20 mM Hepes, pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6mM dextrose) at 230 V and 500 μF using Gene Pulser II (Bio-Rad). Cells were selected with puromycin (1 μg ml−1) and resulting colonies were picked and further expanded. Homologous recombination was verified by PCR as well as Southern blot analysis. For ubiquitous expression of GFP, iPS25Δ1 was electroporated with 1 μg of pPB-UbC.eGFP-neo and 5 μg of pCMV-mPBase using the same conditions as above and selected with G418 (180 μg ml−1). Resulting colonies were picked and further expanded.
Preparation of splinkerettes
Splinkerettes were prepared by annealing Spl-top and Spl-sau (for enzymes generating 5′-GATC protruding ends), Spl-blunt (for enzymes generating blunt ends), or Spl-CG (for enzymes generating 5′-CG protruding ends). Sequences of these oligonucleotides are listed in Supplementary Table 2
. Following heat denaturation at 95 °C for 10 min, annealing was performed by cooling down a mixture of 11 pmol of each strand in 10 mM Tris-HCl (pH7.4) and 5 mM MgCl2
in total volume of 100 μl.
Transposon integration site analysis
Transposon integration sites were determined by Splinkerette PCR 36
. Genomic DNAs of primary iPSCs (0.5 μg) were digested by one of 4-base pair cutters, Mbo
I, and Hin
P1I for 2 hrs in 20 μl. After heat inactivation, 2 μl of the digestion mixture were used for ligation with the corresponding splinkerette adaptors (11pmol) in 20 μl reactions. One μl of the ligation mixture was then subjected to nested-PCR. Primer pairs Spl-P1/PB3-P1 or Spl-P1/PB5-P1 were used for the first PCR. In the second PCR, pairs of Spl-P2/PB3-P2 or Spl-P2/PB5-P2 were used. Finally, PCR products were directly sequenced to determine genomic sequences flanking the piggyBac
terminal repeats. Sequences were analyzed by Blat search on the UCSC genome browser.
Total RNA was extracted by using TriZol reagent (Invitrogen). One μg of total RNA was reverse-transcribed using an oligo(dT) primer by SuperScriptII (Invitrogen), and subjected to PCR using primers listed in Supplementary Table 2
. Quantitative RT-PCR was performed using Platinum SYBR Green qPCR superMix (Invitrogen) on the ABI7900HT sequence detector (Applied Biosystems). Serial dilutions of each RT-PCR product were used to generate a standard curve. Expression levels of individual transcripts were normalized to Gapdh
Bisulphite sequencing was performed by using EpiTect Bisulfite Sequencing kit (Qiagen). Primers used were listed in Supplementary Table 2
Western blot analysis
The 4-factor or 5-factor expression vectors were introduced into 293T cells by using Lipofectamine2000 according to the manufacturer's instructions. Forty-eight hours post transfection, cells were harvested and suspended in LDS sample buffer (Invitrogen) and proteins were separated on 4-15% gradient gels. Protein blots were analyzed using anti-Oct4 (mouse monoclonal, C-10, Santa Cruz), anti-Sox2 (rabbit polyclonal, H-65, Santa Cruz), anti-Klf4 (rabbit polyclonal, H-180, Santa Cruz), anti-cMyc (rabbit polyclonal, ab11917, Abcam), anti-Lin28 (rabbit polyclonal, ab46020, Abcam), or anti-ß-actin antibodies (mouse monoclonal, AC-15, Sigma).
Cells were fixed by 4% paraformaldehyde/PBS for 15 min at room temperature and permeabilized by 0.05% Triton-X100/PBS for 10 min at room temperature, then blocked by 1% BSA/PBS for 1 hr at room temperature. Cells were washed with PBS and incubated with anti-Nanog antibody (1:300, rabbit polyclonal, ab21603, Abcam) and/or anti-Oct4 antibody (1:50, mouse monoclonal, C-10, Santa Cruz) over night at 4°C. After washing with PBS (6 × 10 min), cells were labeled with Alexa488 or 555-conjugated secondary antibodies (Invitrogen) for 1 hr at room temperature. Cells were then washed with PBS (3 × 10 min) and nuclei were counterstained with Toto-3 at 0.5 μM in PBS for 1 hr at room temperature. After final washing with PBS (3 × 10 min), specimens were analyzed using a fluorescence microscope or Radiance2000 confocal microscope (Bio-Rad). The Vectastain ABC kit (Vector laboratory) was used for chromogenic detection according to the manufacturer's instructions.
Teratoma formation and blastocyst injection
Approximately 1 × 106 integration-free iPSCs were injected subcutaneously into dorsal flanks of recipient SCID mice. Tumors were isolated 4 weeks later and subjected to histological analysis. GFP-marked integration-free iPSCs were microinjected into C57Bl/6-Tyr c-Brd/c-Brd blastocysts and embryos were transplanted into CBA×B6F1 pseudopregnant females. In some experiments, embryos were dissected at the indicated time points and observed under the fluorescent stereomicroscope. Chimerism of newborn mice was analyzed by PCR using tail DNAs with GFP primers. All animal studies were carried out at the Wellcome Trust Sanger Institute under the UK Home Office license 80/2020.