SUPPLEMENTARY INFORMATION
Regulation of Somatic Cell Reprogramming through Inducible Mir-302 Expression
Shi-Lung Lin1*, Donald C. Chang1, Chun-Hung Lin2, Shao-Yao Ying3, Davey Leu1 & David T.S. Wu1
1
WJWU & LYNN Institute for Stem Cell Research, Santa Fe Springs, CA 90670, U.S.A.,
2
Taiwan Adventist Hospital, Taipei, Taiwan, R.O.C., and 3Department of Cell & Neurobiology,
Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, U.S.A.
*Correspondence:
[email protected]
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SUPPLEMENTARY INFORMATION Supplementary Materials and Methods Supplementary Figs. S1 to S4 Tables S1 and S2 References Supplementary Materials and Methods Construction of The RGFP Transgene Encoding mir-302s (RGFP-mir-302s). The RGFP-mir-302s transgene was generated as previously reported with minor modifications (Lin and Ying, 2006). The mir-302 familial cluster consists of four parts, including precursor miRNAs (pre-miRNAs) of mir-302a, b, c, and d. Synthetic oligonucleotides (Sigma-Genosys, St. Louis, MO) were listed in Table S1. We first hybridized mir-302a-sense to mir-302a-antisense, mir-302b-sense to mir-302b-antisense, mir-302c-sense to mir-302c-antisense, and mir-302d-sense to mir-302d-antisense, respectively, at 94°C for 2 min, at 70°C for 20 min, and then at 4°C in 1 x PCR buffer. Next, the hybrids (100 pmole each) of mir-302a, mir-302b, mir-302c, and mir-302d were separately digested with MluI/XhoI, XhoI/NdeI, NdeI/XbaI, and XbaI/PvuI restriction enzymes, respectively, at 37°C for 4 hours. The digested hybrids were mixed together and collected with a gel extraction filter (Qiagen, Valencia, CA) in 30 l of autoclaved ddH2O. Immediately after that, the mir-302 cluster was formed by ligation of all four hybrids with T4 DNA ligase (20 units) at 8°C for 16 hours. For insertion into RGFP intron, we mixed an equal amount (1:1) of the mir-302 cluster and a pre-made SpRNAi-RGFP transgene vector from our previous study (Lin et al., 2008), and then digested the mixture with MluI/PvuI restriction enzymes at 37°C for 4 hours. The digested mixture was collected with a gel extraction filter in 30 l of ddH2O and ligated together with T 4 DNA ligase at 8°C for 16 hours. This formed the RGFP-mir-302s transgene, which could be further cleaved out of the vector with XhoI/HindIII digestion. Construction of The Inducible pTet-On-tTS-mir302s Vector. We first modified a Dox-inducible pSingle-tTS-shRNA vector (Clontech, Palo Alto, CA) by replacing its U6 promoter with a TRE-CMV promoter isolated from a pTRE-Tight plasmid (Clontech). Then, the modified vector was digested with XhoI/HindIII restriction enzymes at 37°C for 4 hours, purified by a gel extraction filter in 30 l of ddH2O, and then mixed and ligated with the XhoI/HindIII-cleaved RGFP-mir-302s transgene (1:1) with T4 DNA ligase at 8°C for 16 hours. This formed the inducible pTet-On-tTS-mir302s vector. Given that the pTet-On-tTSmiR302s vector concurrently expressed a tetracycline-controlled regulatory protein (tTS) to control the transcriptional activity of the TRE-CMV promoter, the induction of mir-302 expression was achieved by using just a single vector, which significantly improved the efficiency and convenience of conventional Tet-On gene induction systems. Further, we also modified another Dox-inducible pTet-On-Advanced vector (Clontech) by removing its neomycin-resistant gene (NeoR) with PvuII/NruI digestion and then blunt-end ligation with T 4 DNA ligase at 16°C for 12 hours. This formed a pTet-On-Adv-Neo(–) vector for enhancing mir-302 expression at high Dox concentrations (>5 M). 2
Supplementary Figures
Figure S1. Construct of the inducible pTet-On-tTS-miR302s vector expressing a red fluorescent RGFP transgene encoding mir-302s. (A) Construct of the mir-302 cluster (mir-302s). (B) MicroRNA microarrays showing the specificity of induced mir-302 expression at 6 hours post treatment of 10 M Dox (n = 3, p < 0.01).
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Figure S2. Schematic procedure for mirPS cell generation with electroporation. The inducible mir-302-expressing pTet-On-tTS-miR302s vector (Fig. 1A) was transduced into adult hHFC by electroporation at 300–400 volts for 150 sec in a hypo-osmolar PH buffer (200 l; Eppendorf). In each test, 10 g of the pTet-On-tTS-miR302s vector was used to transfect 200,000 cultured hHFC derived from as few as two human hair follicles (dermal papilla). After doxycycline (Dox)induced expression, the biogenesis of mir-302 relied on the natural intronic miRNA pathway, in which the mir-302 cluster (supplementary Fig. 1A) was co-expressed with the encoded RGFP gene transcripts and further processed into individual mir-302 molecules by RNA splicing enzymes (spliceosomal components) and RNaseIII Dicers (Lin et al., 2008).
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Figure S3. Reprogramming of human hair follicle cells (hHFCs) to mirPS-hHFCs induced by mir-302. (A) Changes of cell morphology and RGFP (red) expression after Dox-induced mir-302 expression in mirPS cells. (B) Time-course of embryoid body formation from a single mirPS cell after limiting dilution. The cell cycle was estimated to be approximately 20–24 hours at the start but gradually shortened after 72 hours. Scale bars = 100 m.
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Figure S4. Pluripotency. Various tissue types were found in teratoma-like cysts derived from mirPS-NT cells, containing all three embryonic germ layers. Tissue markers: skin epidermis with keratin 14 and 16, hair follicle with microphthalmia-associated transcription factor (Mitf) and TRP1, skeleton muscle with M-cadherin and myosin heayy chain (MHC), smooth muscle with actin alpha 2 in smooth muscle aorta (a-SMS, ACTA2) and ED-A fibronectin, endothelium with CD105 and EN4 antigen, gut with mucin 2 (MUC2) and 5B (MUC5B), and gland and glandular epithelium with MUC2, secretory component glycoprotein (SC), glutamic-acid decarboxylase (GAD67) and/or bone morphogenetic protein 11 (BMP11). H&E, histological staining with hematoxylin and eosin. BF, bright field with differential interference contrast.
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Tables Table S1. Name
Sequence
mir-302a-sense
5’-GTCACGCGTT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATAGATCTCT C3’
mir-302a-antisense
5’-GAGAGATCTA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GAACGCGTGA C-3’
mir-302b-sense
5’-ATAGATCTCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTCA TATGA-3’
mir-302b-antisense
5’-TCATATGAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGAGA TCTAT-3’
mir-302c-sense
5’-CCATATGGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3’
mir-302c-antisense
5’-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCCATATGG3’
mir-302d-sense
5’-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGCGATCGC AT-3’
mir-302d-antisense
5’-ATGCGATCGC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3’
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Table S2. Probe
Sequence
Oct3/4
5’-GCAGTGTGGG TTTCGGGCAC TGCAGGAACA AATTCTCCAG GTTGCCTCTC ACTCGGTTCT CGATACTGGT TCGCTTTCTC TTTCGGGCCT GCACGAGGGT TTCTGCTTTG-3’
Sox2
5’-TGCTGTAGGT GGGCGAGCCG TTCATGTAGG TCTGCGAGCT GGTCATGGAG TTGTACTGCA GGGCGCTCAC GTCGTAGCGG TGCATGGGCT GCATCTGCGC TGCGCCGTGC-3’
Nanog
5’-CGTGTGAGGC ATCTCAGCAG AAGACATTTG CAAGGATGGA TAGTTTTCTT CAGGCCCACA AATCACAGGC ATAGGTGAAG ATTCTTTACA GTCGGATGCT TCAAAGCAAG-3’
Lin28
5’-AGGTCCGGTG ACACGGATGG ATTCCAGACC CTTGGCTGAC TTCTTAAAGG TGAACTCCAC TGCCTCACCC TCCTTCAAGC TCCGGAACCC TTCCATGTGC AGCTTACTCT-3’
UTF1
5’-CTGCTGGGCC AGCGCGGCCG ACACGCGGCG GTAGGTGGGC AGGGCCTGGC GGCGGTCCAG GAGCAGCGCG CGCCACACGG CCGGTTGCAG CAGCGTCCCC AGCAGCAGCT-3’
Klf4
5’-CTGCTCGACG GCGACGACGA AGAGGAGGCT GACGCTGACG AGGACACGGT GGCGGCCACT GACTCCGGAG GATGGGTCAG CGAATTGGAG AGAATAAAGT CCAGGTCCAG -3’
FUT3
5’-GAGCCCTAGG GGATCCAGTG GCATCGTCTC GGGACACACG CAGGTAGGAG AAGAAACACA CAGCCACCAG CAGCTGAAAT AGCAGTGCGG CCAGACAGCG GCGCCATGGC-3’
AOF2
5’-CTTCTCTGCT TTGGCATTTC TCTCTTCTTC TGAATAATAC TCATCTTCTG AGAGGTTGGC CAAGCTTTCA TCCATCTCTC TGTACTCTAC CTTCGCCCGC TTGCGCCGGC-3’
MECP-p66
5’-GCCTCATGCC CCTCCATTTT GAGTCGCTTT GCCAGGACAT CATCTCGCTC ATCTGCTGGG TCCAAGCTCC GCTTCAACAG ATTCAAGCGA AGAGCATCTT CTGTCATTCT-3’
MECP2
5’-TGACCCTTCT GATGTCTCTG CTTTGCCTGC CTCTGCGGGC TCAGCAGAGT GGTGGGCTGA TGGCTGCACG GGCTCATGCT TGCCCTCTTT CTCTTCTTTC TTATCTTTCT-3’
CDK2
5’-GTGAATGACA TCCAGCAGCT TGACAATATT AGGATGGTTA AGCTCCTTAA GCAGAGAGAT CTCTCGGATG GCAGTACTGG GCACACCCTC AGTCTCAGTG TCCAGGCGGA-3’
cyclin D1
5’-GACCTCCAGC ATCCAGGTGG CGACGATCTT CCGCATGGAC GGCAGGACCT CCTTCTGCAC ACATTTGAAG TAGGACACCG AGGGCGCGCA GGTCTCCTCC GCCTTCAGCA-3’
cyclin D2
5’-CATGTAGGGT TGGATGTCCT TCTGCACGCA CTTGAAGTAG GAGCACTGCG GAAGGTAGCG CTCCTCGATG GTGAGCAGGT TCTGCAGGAC GCGGTCGTCT CGGAGCAGGT-3’
RGFP
5’-CGAAGGGGTT GCCGTCGCCC TCGCCCTCGC ACTTGAAGTA GTGGCCGTTC ACGGTGCCCT CCATGTACAT CTTGATGCGC ATACTCTCCT TCAGCAGGCC GCTCACCATA-3’
ß-actin
5’-AATGTCACGC ACGATTTCCC GCTCGGCCGT GGTGGTGAAG CTGTAGCCGC GCTCGGTGAG GATCTTCATG AGGTAGTCAG TCAGGTCCCG GCCAGCCAGG TCCAGAGCGA-3’
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References 1.
Lin,S.L. and Ying,S.Y. (2006) Gene silencing in vitro and in vivo using intronic microRNAs. Ying,S.Y. (ed.), MicroRNA protocols. Humana press, Totowa, New Jersey, pp 295–312.
2.
Lin,S.L., Kim,H. and Ying,S.Y. (2008) Intron-mediated RNA interference and microRNA (miRNA). Front. Biosci., 13, 2216-2230.
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