V〇A (12) 特許協力条約に基づいて公開された国際出願 (19
Contents
1. WO 2011 145615 17 PCT JP2011 061311 00502. Mm 2
3. ERBE CH 2 96 EL SE FAA
4. a b
5. 1 2 WO 2011 145615 5 PCT JP2011 061311 001 2 001 3 001 4 001 5 3 4
6. 1 1
7. 1 9 1 6 BLAST WO 2011 145615 6 PCT JP2011 061311 001 6 001 7 001 8 80 90 95 98 9 9 hh OCT4 POU
8. 3 1 1 2 2 0 1 1 2
9. IRES 1 2 1 a NTF 1 mRNA mRNA 2A mRNA 2A 1 2A 5 R 3
10. 1x 104 1x 105ce lls mL ES 1 12 2 10 ES 5 4 0 10 3 5 WO 2011 145615 20 PCT JP2011 061311 0058 0059 Rex 1 Oct4 Fbx 15 NANOG ALP SSEA_3 SSEA_4 AB CG 2 E_ cadherin
11. 1 WO 2011 145615 9 PCT JP2011 061311 0023 0024 0025 0026 RNA mRNA 2 A 2A FMDV 2A F2A A 2A E2A Porcine te schovirus PTV 1 2A P2A Th osea asigna virus 2A T2 A 1 T2A 6 P2A 2A 5 627 638 200 5
12. 1 WO 2011 145615 12 PCT JP2011 061311 gt
13. 0035 2 2 a OCT4 KLF4 SOX2 LIN28 NANOG MYC 3 1 b OCT 4 0036 a OCT4 KLF4 S OX2 LIN28 NANOG MYC 3 a 3 4 5 6 a
14. 1 0 WO 2011 145615 35 PCT JP2011 061311 0104 10 amp 58 B AJ BOAT n 3 cells pDON5 mOct4 pDON5 mKIfA T A 7 33 0 0796 pDON5 mSox2 pDON5 me Myc B p DON5 mOKSM 0 67 o 0 01 DON5 mOKSM c 30 33 3x10 1 01 pDON5 mOct4 D pDON5 mOKSMO 0 00 1x10 0 00 pDON5 mOKSMO E 1 33 3 103 0 0496 pDON5 raOct4 F pDON5 mO IR KSM 8 33 lxIO 4 0 0896 pDONS mO IR KSM G 35 67 3x1 1 1996 pDoN5 moct4 pDON5 mO R KSM H 6 33 4 0 0696 pDON5 mKI f 4 pDON5 m0 IR KSM 4 33 4 0 0496 pDON5 mSox2 pDON5 m0 IR KSM J 3 33 lxIO 4 0 03 pDONSic Myc 1 0 A B C 4 4 2A mOct 4
15. 1 SB a WO 2011 145615 14 PCT JP2011 061311 0040 0041 0042 codon frequency 2 5 2A 2 1 a RES 1 mRNA
16. 96 17073 BILLY hys 1 2 1 WO 2011 145615 18 PCT JP2011
17. 10 F_DMEM 2 4 1 1mL 3 2 C 2 000xg 2 0099 1 5 HSA Human Se rum Albumin PBS 1 4 x 104cells mL 10F_DMEM 3 MEF 1mL 3 7 C CO 1 3 0100 3 6 1mg mL 1 4 C 1 2 gt g mL C SNL76 7 D S 1 1x 106ce l ls
18. ES HS BE in d uc ed piuripotent stem cell iPS 1 1 OCT4 SOX2 c_ MYORON 4 4 WO 2011 145615 2 PCT JP2011 061311 0006 f iPS OCT4 SOX2 NAN OG L IN2 8 4 iPS EF 2
19. JB 9 9 3 7 1 8 WO 2011 145615 PCT JP2011 061311 1 Aexl Oct3 4 Endo Fbx15 Nanog Gapdh 2 3 WO 2011 145615 PCT JP2011 061311 3 Mesodenn Endoderm tfriodernm 3 3 PCT JP2011 061311 WO 2011 145615 4 uuu uuu SHOU INTERNATIONAL SEARCH REPORT A CLASSIFICATION OF SUBJECT MATTER C12N1 509 2006 01 i 2 2006 01 n CO7K1 90 0 2006 0 2006 01 i 01 n According to International Patent Classification FIELDS SEARCHED E International applic
20. a b a b WO 2011 145615 19 PCT JP2011 061311 0054 0055 0056 0057
21. K IY 1 I amp ROS OME x 03 3581 1101 3448 2009575 PCT ZJP20117 061311 C Wo 2010 004989 A1 2010 14 0021 0023 0051 0062 amp EP 2295538 A1 PCT 1SA 210 2 200957
22. OCT4 KAR RefSeq Acc No NM 00 2 701 4 OCT 4 13 1405 2484 OCT 4 Kee t RefSeq Acc No NM O 13633 OCT 4 10 13 96 245 1 OCT 4 mRNA RNA m i RNA m iR_470 m iRNA OCT 4 m iRNA OCT 4
23. iPS 0 0 2 lt PS OCT4 KLF4 SOX2 L IN28 NANOG 5 iPS iPS MUIR iPS 3 iPS iP S
24. mOct 4 iPS 105 5 PS 4 G pDON5 IR_KSM pDON5 mO ct 4 iPS iPS 0106 1 iPS 4 1 2 3 iPS io 2 2 1 iPS 52 61 OLYMPUS X iPS 0 25 trypsin EDTA 100 9 6 3 7 C 5 iPS SNL76 7 1 5 4x 1 04ce ls 24
25. OCT 4 KLF4 SOX2 LIN28 NANOG MYC 6 MAR Z 2 5 OCT4 KLF4 SOX2 LIN28 NA NOG MYC 1 3 1 2 4 1 OCT4 KLF4 SOX2 LIN28 NANOG MYC 3 Ff 1 2 OCT 4 BREL 5 1 3 NARA IRES int ernal ribosomal entry site Hz WO 2011 145615 4 PCT JP2011 061311 001 0 001 1 4 6 1
26. iPS ES FBS 1 0 iPS iPS WO 2011 145615 37 PCT JP2011 061311 01 08 01 09 01 10 01 11 01 12 6 ES 5 iPS ES 7 RT PCR ES 1 RNA 6 i PS Fa stPur e RNA Kit To talR NA K i t Tota 1IRNA 2 cDNA 6 1 Tot alIRNA cDNA Pr imeScr ipt R
27. DDON5 3XO0 D IDON5 mOKSM amp T 0 4 pDON5 mc My cC EOpDONS5 m Oc t 4 DNA 7 pDON5_mOKSM 10 To 7 DDON5 mOKSM mOct4 T2A1 mK1f4 2 2 T2A3 mc Myc pDON5 mOKSMO mOct4 TZA1 mK1f4 mSox2 T2A3 mc Myc T2A4 mOct4 6 iPS pDON5_mO_ R K SM 5 _ pDON5_mOKSM 1 T2A1 1IRES2 pDON5 mO 1IR KSM plIRES2 Zs Green PCR IRES2 In Fusion Advantage PCR pDON5_mO_ _ KSM 2 pDON5_mO K S M DNA
28. AUP KTS WO 2011 145615 11 PCT JP2011 061311 0031 0032 0033 0034 Codon frequency 2 5 T 2 A 7 P 2 A 1 1
29. 2 3 4 5 P2A 6 7 T2A1 T2A2 T2A3 T2A4 P2A DNA 0062 DNA 2A F DNA PCR 2A DNA 5 X 1 DNA pDON 5 DNA pDON5_hOKS pDON5_hOKSL pDON5 hOKS LN P2A pDON5_hOKSLN P2A P2A T2A4 pDON5 hOK SLN T2A 0063 X1 pDON5 hOKS hOCT4 T2A1 hKLF4 T2A2 hSOX2 pDON5 hOKSL hOCT4 T2A1 hKLF4 hSOX2 T2A3 hLIN28 pDON5 hOKSLN P2A hOCT4 T2A1 hKLF4 T2A2 hSOX2 T2A3 hLIN28 P2A hNANOG pDON5 hOK
30. 1 1 1 3 B 1 2 3 2 SCGM L onza PGM_ 2 Lonza 2 Te WO 2011 145615 28 PCT JP2011 061311 0081 0082 0083 0084 2 6 1 4 E 7 2 1 3 iPS 2 8 1 5 iPS 5
31. ES 14 15 STO C ES 1 2 ES iPS
32. 3 5 2 OCT4 KLF4 IRES 4 5 7 4 6 1 2 8 1 4 9 1 4 10 9 1 1 9 12 3 7
33. pDON5_hOKS T2A4 hc_MYC DNA pDON5_ hOKSM DNA 9 pDON5_ 0O 3 HDON5 DNA he _ MYC DNA pDON5_hc_M YC pDON5_ hOKSM 13 hOCT 4 DNA pDON 5 DNA pDON5_ hOCT4 pDON5_ hOCT 4 1 4 WO 2011 145615 32 PCT JP2011 061311 0093 0094 0095 0096 3 9 5 XAERDERSIUSDNAZTZJ ONE pDONS hOKS OCT4 T2A1 hKLF4 T2A2 hSO0X2 pDONS hOKSLN hOCT4 T2A1 hKLF4 hSOX2 T2A3 hLIN28 T2A4 hNANOG pDON5 hOKSM nOCT4 T2A1 hKLF4 T2A2 hSOX2 T2A4 hc MYC pDON5 hOCT4 hOCT4 8 1 G3T hi
34. 4 1 6 c _ 3 7 C C0 WO 2011 145615 24 PCT JP2011 061311 0069 0070 0071 24 5001 10 L T rans IT 293 5 2 g pGP 1 gt g E_Ampho 2 g 1 2 15 G3Thi 24 4mL 10F DMEM 2 4 ww 0 4 5 um 8 0 C 3 7 C 2 10F D
35. c _ MYC 13 50 44 6360 c MY C RBM RefSeq A cc No NM 0 10 849 N MYC Mat RefSeq Acc NM 008709 L MYC NAME RefSeq Acc N NM 0 0 8506 c_ MYC 1 0 5077 6393 BOOM AF 2 2A 2A 2A 2A
36. 1 pDON5_ hOKS pDON5 hOKSL pDON5 hOKSLN P2A T 2A 4 c MYC pDON5_hOKSM pDON5 hOKSLM pDO N5 hOKSLNM DNA 2 1 pDON5 _ hc_MYC 352 B DNA pDON5 hOKSM hOCT4 T2A1 hKLF4 T2A2 hSOX2 4 hc MYC pDON5 hOKSLM hOCT4 2 hKLF4 T2A2 hSOX2 hLIN28 2 4 hc MYC pDONS hOKSLNM hOCT4 T2A1 hKLP4 2 3 hLIN28 P2A hNANOG T2A4 hc MYC DNA 4 pDON5_hOKSL NM 8 3 1 G3Thi 5x 105cells mL 10F DMEM 10 50U mL 5Omg mL KLZ R D MEM
37. 3 ARB Ectoderm BRE Endoderm PRR Mesoderm 4 G pDON5 mO IR KSM pDON5 mOct4 iPS 123 9 OCT4 OCT4 iPS hOCT4 hSOX2 hKLF4 hc_MYC 4 hc_ MYC hOCT4 hSOX2 hKLF4 hL IN28 hNANOG 5 124 1 MORE 4 1 125 2 8 12 A D 10 F DMEM 3 0
38. ES S SEA 1 8 in vivo iPS 5 iPS ES SC1D 1 iPS WO 2011 145615 40 PCT JP2011 061311 iPS 0 25 Trypsin EDTA 5x 106cel ls mL 121 2 iPS SC1D 8 1 iPS SC1D 5 x 105ce lls 100 L 122 3 iPS 5 SC1D Z EL TISSUE MOUNT 8 0 C HE
39. PCR 1 0 hKLF4 1 5 kbp DNA hSOX2 1k bp D NA hc_MYC 1 3 kbp DNA RefSeq Acc No NM 002701 4 hOCT4 NM 024674 4 hL IN28 NMO 24865 2 hNANOG hOCT4 1 1k bp DNA hL IN28 0 7 kbp DNA h NANOG 1 k bp DNA Thosea asigna Vtrus Porcine tesc hov irus_ 1 2A T2A P2A lt Nature Biotechnol ogy 22 5 589 94 2004 T 2A 1 T2A 4 T 2 WO 2011 145615 22 PCT JP2011 061311 A1 T2A2 T2A3 T2A4
40. M RES internal ribosoma ll entry s i t e MAR A UG Trends Biochem Sci 15 12 4 77 8 3 1990 1RES A mRNA BRE MRNADFOMRORRE US
41. mRNA 1IRES Trends Biochem Sc ii 15 12 4 77 83 1990 EMCV BR FMDV AR HCV C BB RES BiP VEGF AR E Mol Cell Biol 18 3 8115 86178 61908 1998 2 e IF4G TF ID HAP4 mR NA 1IRES 2 1 a
42. KLF4 KLF4 KAR I Re f Seq Acc NM 004235 3 K L F 4 13 2 5 5 1 3 9 6 0 K LF4 KAR RefSeq Acc No NM 0 10 637 KLF4 10 2 5 18 3966 SOX2 Sox SRY_ related HMG box WO 2011 145615 7 PCT JP2011 061311 001 9 0020 0021 DNA HMG Jn SOX2 MASE RefSeq Acc No NMO 03106 2 SOX 2 KMAR It 13 4027 4977
43. SOX2 KMAR It RefSeq Acc NM 0O 11443 SOX2 10 40 5 1 5007 SOX2 OCT 4 m iR 134 m iRNA SOX2 LIN28 1let_ 7miRNA R NA L IN28 MER RefSeq Acc NM 02467 4 4 L IN28 12 5044 5670 L N2 8 Re f S eq Acc NM 145833 NANOG
44. 4 6 1m g mL in L 3 7 cc s o 1 2 g mL c Ss NL7e 7 0s 2 s x 1o sc e is 3 7xc co 1 3 e 1oF DMEM 2ooooce 11s Z 7 g s js 0 D MEM F 12 20 knockout Ser um Replacement 4 ng mL human basic FGF CALBIOCHEM 1xNo n essential amino acids solution L onzat 2mM L Giuthamine Lonza 11ouM 2 sou mL s om gmL 2mL
45. F 1 RES WA BAI RES 1 5 1 2 2 b OCT4 WO 2011 145615 15 PCT JP2011 061311 0043 0044 0045 MAA 8 100mRNACUC ES mRNA OCT4 b a mRNA 2 a b
46. 3 SARA 1IRES internal ribosoma entry site 4 1 3 5 2 O CT4 KLF4 RESHMA et 4 5 4 6 1 2 1 4 WO 2011 145615 9 10 11 12 45 PCT JP2011 061311 1 4
47. 1mL L 4 C PBS 1 4 C 2 1 1 1 3 A B C 1mL 32 C 2 0 0 0xXg 2 1 5 PBS 1 4 x 104ce ls mL 10F DMEM Lonza 1mL 3 7 C CO GHOAB 2 1 5 PBS 4 C 1 12
48. BK WO 2011 145615 21 PCT JP2011 061311 0060 0061 1 2A PS pDON5_hO KS pDON5_hOKSL pDON5 hOKSN pDON5 h OKSLN P2A pDON5 hOKS LN T 2A RefSeq Acc No NMO 04235 3 hKL F 4 003106 2 hSOX2 NM 002467 3 hc MY Cili z f HumanBrainPolyA RNA cDNA Pr imeSTAR MAX PreMix HS DNA polymeras e
49. 4x104cells mL 10F_DMEM 1mL 1 A B C 8 g mL WO 2011 145615 26 PCT JP2011 061311 0074 0075 1mL GB HH 3 2 2 1 1oF puEu ROR 1 1 1 2 10F DpMEM 1mL
50. NANOG KRM RefSeq Acc N o 024865 2 NANOG k amp 12 5743 6657 NANOG MER RefSeq Acc 13275 5 MYC WO 2011 145615 8 PCT JP2011 061311 0022 c MYC N MY C L MYC 47 L Cell Sc E 119 208 216 20 0 6 c MY C RefSe q Acc No NM 002467 N_MYC eM RefSeq Acc No NM 005378 L MYC NAMIE RefSeq Acc No NM 005376 c_ MYC RefSeq Acc NM 002467 3
51. PB S 2 4 10 PBS 2 2 1 BSA Bovine Serum Albumin PBS SSEA_ 1 XIA IgM R amp D 1 BSA PBS 50 4 C 3 PBS 3 A lexa48 8 IgM 1 0 0 0 4 C PBS 3 2 2 PS
52. D 4 mOct 4 2A Oct 4 F mOct4 mK1I1f4 2A RES 2A B 8 1I RES 1IRES mnKIf4 mSox2 mc WO 2011 145615 36 PCT JP2011 061311 My c iPS F mOct4 G mOct 4 mK 1If4 DNA RES iPS
53. WO 2011 145615 10 PCT JP2011 061311 0027 0028 0029 0030 1 6 1 OCT4 KLF4 SOX2 LIN28 N ANOG MYC 6 BI Of 6 5 OCT4 KLF 4 SOX2 L IN28 NANOG MYC 1 1
54. 3 27 2 000xXg 2 WO 2011 145615 41 PCT JP2011 061311 01 26 01 27 01 28 01 29 01 30 1 5 HSA PBS 1 4 x 104ce ls mL 10F DMEM 1mL 3 7 C C0 S0 HH BB 1 3 3 6 1mg mL 1 4 C 1 2 gt g mL C SNL76 7 1 1x 106cells 3 7 0 9 2 6 5X 103ce ls
55. a b MAA a b WO 2011 145615 16 PCT JP2011 061311 0046 0047 0048 0049
56. 8 1 1 WO 2011 145615 31 PCT JP2011 061311 0090 0091 0092 8 5 DNA pDON5 mO TR KSM mOct4 IRES2 mKif4 mSox2 T2A3 mc Myc 7 2A iPS pD ON5 hOKS pDON5_hOKSLN pDON5_hOKSM pDON5_ hOCT 4 1 DNA 2A DNA PCR 2 A DNA 5 9 DNA pDON 5 D NA pDON5_hO 3 HDON5 hOKSLN Lz pDON5 hOKSLN 12 In Fus ion Advantage PCR
57. iPS 1 Ce 1 13 1 86 1 8728 2007 2 Sc ience 3 18 19 17 19 205 20075 3 Sc ience 322 949 953 BH 2008 WO 2011 145615 3 PCT JP2011 061311 0007 0008 0009 ES 1
58. 8 0 SC 3 Mouse embryonic fi broblast MEF i PS MEF Takahashi K WO 2011 145615 33 PCT JP2011 061311 Nature Protocol 28 3081 30898 20075 0097 4 iPS 1 MOREL 12 2 5 g mL PBS 1mL 4 C 1mL PBS 2 4 C 098g 2 8 10 A J
59. 3 WO 2011 145615 34 PCT JP2011 061311 01 0 1 01 02 01 03 7 C CO gt 4 2 4 RFA B D F H 1 Jl amp 5x 103c el ls mL lor DMEM C E G 1 5x 103celils mL 10F_ DMEM 2 mL ES 8 5 DMEM 1 5 1 x LONzA 1mM LONZAtER 2mM L LoNzA 2 _ 2m L 2 8 COM 1 2 4 iPS 2 8 ips Es
60. WO 2011 145615 29 PCT JP2011 061311 0085 0086 1 0 mOct 4 1 1kbp DNA mKIlf 4 1 5 kbp DNA mSo x 2 1 0 k bp D NA mc_Myc 1 3 kbp DNA DNA p DON 5 DNA pDON5 mOct4 pDON5 mKIf4 pDON 5 _mSox2 pDON5_mc_Myc pDO N5 mOct 4 9 6 Mouse ES Mouse Spleen 1 f 4 mc Myc 5 2A i PS p DON5 mOKS pDON5_mOKSM DDON5 mOKSM O 4 DNA 7 1
61. ES Ce 1 12 6 6 63 676 200 6 2 4 2 14 N CB N C cDNA PCR DNA
62. 3 5 3 4 5 2 0 ES m RNA iPS 1 8 1 1 MEF ES iPS ES PS ES WO 2011 145615 39 PCT JP2011 061311 01 15 01 16 01 1 7 01 18 01 19 01 20 7 SSEA 1 Stage specific embryon ic antigen 1 5 iPS ES 1 iPS 2 4 iPS
63. 5 x 105ce lls mL 10F DMEM 10 Hyc lone 1 co KLZ D MEM 4mL 6 cm 3 7 C C0 2 4 BASE 500 L OPTI MEM 1 0 LOT rans IT 293 5 2g pGP 1 gt g pE Eco pE Ampho 2 g 4 7 15 G 3 T hi 2 4 4mL 10F DM E M 2 4 XL 0 4 5 m
64. 126 663 67 6 2006 13 1 86 1 872 2007 E s GFP DNA 500 1000cells 4 1 2 SHE SE
65. mRNA mRNA 2A 1 mRNA 2A 1 2A 5 3 R 1 1 GRE 1
66. 5 i PS 2 Blea ro Tav s 111 4 5 pDON5_ hOKSLNM 4 iPS pDON5_mOct 4 pDON5 mKIf4 pDON5 mSox2 pDON5 mc My c RefSeq Acc No NM 013633 2 mOct 4 Z NM 0 10 637 3 mKIf4 NM 011443 3 mSox 2 NMO 10849 4 mc Myc MouseES DS MouseSpleen TotaIlIRNA cCDNA Pr imeSTAR GXL DNA Polymerase PCR 6
67. 2 1 Y Wo 2010 008054 A1 2010 01 21 0062 amp CA 2731007 A iW c IA IE L IO IP A 11 SA JP 100 8915 4 3 PCT 1SA 2 10 2 0032 0036 0045 1
68. 2A DNA PCR MARRU 2A DNA 5 mOct4 T2A1 mKIf4 T2A2 mSox2 DNA DNA pD ON 5 DNA pDON5 mo KS DNA 7 ln Fus ion Advantage PCR WO 2011 145615 30 PCT JP2011 061311 0087 0088 0089 YR pDON5 gO 3 2A 3 mc_Myc DNA pDON5_mOKSM pDON5 0 3 MM 2 A 4 mO ct 4 DNA pDON5_mOKSMO
69. 2 s COM 2 WO 2011 145615 27 PCT JP2011 061311 0076 0077 0078 0079 0080 5 iPS 26 TRACP amp ALP doub le stain Kit AP ES iPS 4 4 i PS 1 522 475 165 Jas 18 3 4 2 6 e 142 143 se 184 156 26 4 pDON5_hOKSLNM pDONS5 hOKSLN T2A A 2 iPS Lo nza Lonza
70. 71 lt BB BG BH BR BW BY BZ CA CH CL CN CO AkARA BIO INC JP JP CR CU CZ DE DK DM DO DZ EC EE EG ES T 502193 4 GB GD GE GH GM GT HN HR HU ID IL IN IS Shiga JP JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PG PH A PL PT RO RS RU SC SD SE SG SK SL SM ST ENOKI Tatsuji JP JP T 5202193 SV SY TH TJ TM TN TR TT TZ UA UG US UZ A 1 VC VN ZA ZM ZW shi JP NisHIE Toshik JP JP en Hz T Aa 3 6 RTOSURY STORAOLMR 1 shiga Op AB 8b TB ARPO BW GH GM LR LS MW 54 Title NUCLEIC ACID FOR PRODUCTION OF PLURIPOTENT STEM CELL 64 E24 h ks e KIES xi SK gt AMAA TAL T2A2 hOKSL Er XC 7 xm ELS aac 2A1 T2A2 T2A3 hOKSLN P24 AAAA 2A1 T2A2 TAS 2 MOKSIN28 CRE PED EE ECCE Cy gt TAL
71. BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS Ir LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR OAPI 5 2 a BF BJ CF CG CM GA GN GQ GW ML MR NE SN TD TG O CT4 KLF4 SOX2 L IN28 NANOG MYC 6 a RES OCT4 KLF4 SOX2 LIN28 N ANOG MYC 6 R b OCT 4 WO 2011 145615 1 PCT JP2011 061311 0
72. T2A2 T2A3 T2A4 hGKsM TTA m KiEX ECE SEER ae 1 T2A2 T2A4 hDKSLM CNTA gt al HES ps TIT ETP ELEME AMA rai 1242 1293 284 bOKSLhN mO IR KSM DETA KIFE gt FT 3 XII Aa en M22 4203 57 Abstract The purposes of the present invention are to provide a method for efficiently producing pluripotent stem cells and to promote the study and use of the pluripotent stem cells Specifically disclosed are a nucleic acid which contains coding regions of genes that respectively encode six nuclear reprogramming factors OCT4 KLF4 SOX2 LIN28 NANOG and a protein belong ing to the MYC family said coding regions being linked to one another through sequences encoding self cleaving peptides a set of a a nucleic acid which contains regions that are selected fiOm among coding regions of genes that respectively encode six clear reprogramming factors OCT4 KLF4 SOX2 LIN28 NANOG and a protein belonging to the MYC family said regions be ing linked to one another through IRES sequences or sequences encoding self cleaving peptides and b another nucleic acid wmch contains a coding region of a gene that encodes OCT4 and a set of vectors which respectively comprise the nucleic acids 67 WO 2011 145615 MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM a we ce a ap oe amp 858 21 AL AT BE
73. mL 10F DMEM CDRA 2 mL ES 80 DMEM F12 20 1 x 2mM L 100 gt M 2 4ng mL bFGF 2mL L 2 8 COM 1 2 4 iPS 2 8 iPS 12 WO 2011 145615 42 PCT JP2011 061311 12 SoS nN 0 n 3 96 A pD ON5 hOKSM 22 7 0 06 B ONS OOM 266 0 0 67 pDON5 hOCT4 C pD ON5 hOKSLN 15 3 0 04 pD ON5 hOKSLN P m pDON5 hOCT4 12 hOCT4 hSOX2 hKLF4 C 4 hc MYC hOCT4 hSOX2 hKLF4 hLIN28 hNANOG 5 O
74. oouenbes 9 109 9 90 10 1 194 6 ON GI 03S ON 03 ON GI 035 ON GI 035 ON QI 03S 03 ON QI 035 ON QI 03S SI9SrI IT0C OM WO 2011 145615 44 PCT JP2011 061311 1 2 3 4 5 6 7 8 OC T4 KLF4 SOX2 LIN28 NANOG MYC 6 5 OCT4 KLF4 SOX2 LIN28 N ANOG MYC 1 1 2 1 OCT4 KLF4 SOX2 LIN28 NANOG MYC 3 1 2 OCT 4 1
75. 001 0002 0003 0004 0005 H pluri potent stem cell BERR embryonic stem ce ES LA UZ 5 k NE EI LTE ES
76. 061311 0051 0052 0053 V DEA E 9 7 183 18 I fh
77. 2011 145615 1 12 7 Oe Ae TON CMOR NNU ON VNV N DTN NDD ONT 43 10 mm 4x BE 2011 11 24 4 11 2011 et WO 2011 145615 A1 6 EE ZR MARUI Takahiro JP JP T 5202103 C12N 15 09 2006 01 A61P 43 00 2006 01 4 B15 C12N 5 10 2006 01 CO7K 19 00 2006 01 shiga uP TAkAsHiMA A61K 35 12 2006 01 Fuyuko JP JP T 5202198 e PCTJP201 1 0613 11 EUM S Shiga p i Junichi JP JP 2 204 1 5 17 Fl 17 05 201 1 5202193 4 a 25 Shiga op S 4 CANAKA Mitsuo etal BAR 1 3 30 7 amp 1M P Osaka P 2010 1 43806 2010 fF 5 A 18 H 18 05 2010 Jp 2010 2sg192 2010 12 27 27 12 2010 JP d Metam WD H AE AG AL AM AO AT AU AZ BA
78. CT 4 0133 seq IDNo 1 T2A peptide fragment 01 3 1 hc MY 01 32 sea D no 2 Nucleotide sequence T2A1 DNA fragment sEQ ID no 3 Nucleotide sequence T2A2 DNA fragment seQ IDno 4 Nucleotide sequence T2A3 DNA fragment of of of sea ID No 5 Nuc leotide sequence of T2A4 DNA fragment sea D uo 6 P2A peptide fragment sea IDno 7 Nucleotide sequence of P2A DNA fragment SsEQ ID uo 8 Nucleotide sequence Of pDON5 hokSLNM insertion fragment sea D no 9 Nucleotide sequence of pDON5 m0ct4 sea ID No 10 Nucleotide sequence Of pDON5 m0KSM ow yt cep pz eui 4 oul ui 47 9U 4 EM QU dd GIXd z Jeui4d qj G Xqu 6 Ou y bl 8 19 q4 Opusy DOW 1911 14 qu Xxeyu 9L 1914 14 gq4 xeyu G 9 c oec uM c qd oouenbes 9p 309 9nwN eI i NISX0u sNoQd jo ae yA
79. MEM 3 06 ER L OCT4 SOX2 Encep ha lomyocarditis virus IRTS inter nal Ribosome Entry Site pDON 5 OCT3 4 SOX2 LIN28 NANOG IRES pDON 5 LIN 28 NANOG KLF4 pDO N 5 KLF4 Human iPS Cell Generat ion Vector Set 10 3 WO 2011 145615 25 PCT JP2011 061311 0072 0073 3 DOR oeo pDONS hOKSLN T2A pDONS5 hOKSLNM Se pDON 5 OCT3 44 SOX2 pDON 5 LIN28 NANOG C pDON 5 KLF4 1 iPS 1 12 20 gmL PBS
80. OC T4 KLF4 OCT4 KLF4 SOX2 MYC OCT4 KLF4 SOX2 LIN28 NANOG MAR Z WO 2011 145615 13 PCT JP2011 061311 0037 0038 0039 a 5 OCT4 KLF4 5 OCT4 KLF4 SOX2 MYC OCT 4 KLF4 SOX2 L IN28 NANOG 2 1 a 1I RES
81. SLN T2A hOCT4 T2A1 hKLF4 T2A2 hSOXZ 5 hLINZS T2A4 hNANOG 0064 2 iPS pDON5_hc_MYC 5 DNA 2A PS pDON5_hOKSM pDON5 hOKSLM pDON5 hOKSLNM E RefSeq Acc No NM 002467 3 c MY F H umanBrainPolyA RNA cDNA c MYC P imeSTAR HS DNA Polymerase WO 2011 145615 23 PCT JP2011 061311 0065 0066 0067 0068 PCR 1 0907 2 1 3 kb c MYC pD ON 5 pDON5 hc MY C In Fusion Advantage PCR
82. T reagen t Kit Perfect RealTime SYBR Green Assay T o t a I RNA 200ng TaKaRa POR Thermal Cycler Dice Gradient i 3 7 C 15 85 C 5 cDNA 3 PCR 1 1 DNA WO 2011 145615 38 PCT JP2011 061311 01 13 01 14 11 i ms mFbx15 RP F anog 6 2 cDNA TotaIRNA 10 ng PCR 5 gt L 5xPrimeSTA R GXL Buffer Z24uLOdNTP Mixture 2 5m M each 5pmo 1 5 pmo 1 0 5 LOPrimeSTAR G XL DNA Polymerase 1 25U EMA RAR 2 5 L T aKaRa PCR Thermal Cycler Dice Gradient 98 C 10 60 C 15 68 C 15 1 Fb x 15
83. ation No PCT JP2011 061311 A61K351 2 2006 01 n Ae61PA430 0 IPC or to both national classification and IPC Minimum documentation searched classification system followed by classification symbols C12N1 5 09 C12N5 10 A61K3 5 12 A61P43 00 C07K19 00 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Jitsuyo Shinan Koho Kokai 1922 1 1 971 2011 996 Jitsuyo Shinan Koho Electronic data base consulted during the international JST Plus JME DPlu s JST7580 JDreaml c DOCUMENTS CONSIDERED TOBERELEVANT eee CONSIDERED TO BE RELEVANT Category wo 2010 030003 AI Ltd 18 March claims 0036 Fami y 2010 paragraph 0045 none 18 03 2010 s 0022 example s 1 WO 2010 008054 AI 21 2010 paragraph 0062 amp CA 2731007 A Dnave 01 January 21 2010 WO 2010 004989 AI 14 January 2010 paragraph S 190233 amp EP 2295538 AI Takara Bi 14 01 2010 to 0023 L Further documents are listed in the continuation of Box C Special categories of cited documents document defining the general state of the art which to be of particular relevance is not considered earlier application or patent but published on or after the international filing date is or other document which may throw doubts on priority claim s or which cited to establish the publication date o
84. f another citation special reason as specified document referring to an oral disclosure use exhibnion or other means document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international 11 2011 11 08 search Augu st 11 Name and mailing address of the ISA Japane se Patent Office Facsimile No Form PCT ISA 210 second sheet July 2009 Jitsuyo Toroku Shinan Toroku Koho Koho 1996 2011 Jitsuyo Shinan 1994 2011 search name of data base and where practicable search terms used PubMed 0023 2 Corp Citation of document with indication where appropriate of the relevant passages Relevant to claim No Kyowa Hakko Kirin Co 0032 1 to fig e Inc e 0051 to 0062 LO See patent family annex T later document published after the international filing date or priority date and not in conflict with the application but cited to understand the principle or theory underlying tiie invention X document of particular relevance the claimed considered novel or cannot be considered step when the document istaken alone invention cannot be to involve an inventive Y document of particular relevance the claimed invention cannot be considered to involve an inventive step when the document is combined with one or more other such documents such combination being obvio
85. us to a person skilled in the art document member of the same patent family Date of mailing of the international 23 Augu 2011 search report st 23 08 11 Authorized officer Telephone No A Int Cl C12N15 09 2006 01 i C07K19 00 2006 01 n I PC B Int Cl C12N15709 C12N5 10 A61K35 12 45 00 JSTPlus JMEDPlus JST7580 jDreamll PubMed C OR C12N5 10 2006 01 i A61K35 12 PCT J P20117 061311 2006 01 n A61P43 00 2006 01 n C07K19 00 2010 03 18 0023 X Wo 2010 030003 A1 Y 00221
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