能書単頁9[1].5(2)

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1 ips CytoTune -ips ver.1.0 1

2 I CytoTune ips 3 II III CytoTune ips ips ) 2) ips 8 ips 8 SeV 9 SeV 10 IV Q&A 11 V VI

3 I CytoTune ips CytoTune ips 4 OCT3/4SOX2 KLF4c MYCSeV ips SeV ips SeV ips II. SeV 1 RNA 2 naïve T SeV RNA Okano, S. et al. Gene Ther, 10, (2003). 3

4 Sendai virus, SeV 1 SeV 1950 SeV HVJHemagglutinating Virus of Japan 1 RNA 15, nm 3 Nucleocapsid Protein, NPRNA Phosphoprotein, P Matrix protein, M (Fusion protein, F)/ Hemagglutinin Neuraminidase, HNRNA Large protein, L 6 4

5 SeV NPPMFHNL F SeV SeV F SeV/TS F SeV/TS15 F DNA SeV RNA SeV US 6,723,532US 6,645,760US 7,101,685US 7,442,544US 7,226,786US 6,746,860CN ZL CN ZL CN ZL HK HK EP EP KR KR KR CA

6 IIICytoTune ips ips A OCT3/4 SeV/TSΔF 100 µl 3 3 x 10 6 CIU / 100 µl B SOX2 SeV/TSΔF 100 µl 3 3 x 10 6 CIU / 100µL C KLF4 SeV/TSΔF 100 µl 3 3 x 10 6 CIU / 100µL D c MYC(HNL) SeV/TS15ΔF 100 µl 3 3 x 10 6 CIU / 100µL BJ 1x GenBank Accession No. OCT3/4NM_ SOX2: NM_ KLF4: BC c MYC: K C 1 6

7 CO 2 (Φ100 mm 6 12 ) (15 ml) (5 ml10 ml) (200 µl1000 µl) IVF Cat. No. H , Cat. No. H 9570, (D MEM ) Basic fibroblast growth factorbfgf: human recombinant ES (4ng/mL bfgf ) ROCK :Y % ( C MEF) ES ES 0.25% EDTA Fetal Bovine Serum FBS PBS anti Sendai Virus Cat. No. PD029, MBL 7

8 ips BJ ATCC No. CRL2522 ips ips 1. BJ 2 5x10 5 cells/well CO 2 37C5% CO C CytoTune ips 37 C ml 10% FBS/D MEM 2 ml 4 ADOCT3/4SOX2KLF4c MYC 10 6 MOI=3 5 ml BJ 4 CytoTune ips 6. CO 2 37C5% CO % FBS/D MEM 2 ml/well 8. CO 2 37C5% CO 56 10% FBS/D MEM * cells/100mm dish MEF 10% FBS/D MEM *0.1% 4 ml/100mm dish 1 ml/well 612 well 37C PBS 1 PBS % EDTA 500 µl/well 10% FBS/D MEMiPS 8

9 cells/100mm dish RT PCR SeV 12. CO 2 37C5% CO ES 37C3% CO 2 HEPES ES 5% cells/well cells/well MEF IVF 10µM ROCK ES 16. CO 2 37 C3% CO 2 HEPES ES 5% ES ES/ ips SeV ips BJ SeV ips 1. ips anti Sendai Virus 2. SeV 3. SeV RT PCR SeV RT PCR 4. anti Sendai Virus ips V 9

10 [] anti Sendai Virus 12 ips PBS 1 ml 10NWAKO 5 PBS µl anti Sendai Virus 0.1% TritonX 100/ PBS 1/ C 1 PBS µl anti rabbit IgG 0.1% TritonX 100/ PBS 1/ C 1 PBS 3 Transgene SeV RT PCR ips RNA 10 RT SeV RNA RT PCR PCR 95 C 30 sec 55 C 30 sec 72 C 30 sec % PCR 10

11 Transgene SeV RT PCR transgene Forward Reverse product size OCT3/4 CCCGAAAGAGAAAGCGAACCAG AATGTATCGAAGGTGCTCAA* 483 bp SOX2 ACAAGAGAAAAAACATGTATGG* ATGCGCTGGTTCACGCCCGCGCCCAGG 529 bp KLF4 ACAAGAGAAAAAACATGTATGG* CGCGCTGGCAGGGCCGCTGCTCGAC 591 bp c MYC(HNL) TAACTGACTAGCAGGCTTGTCG* TCCACATACAGTCCTGGATGATGATG 532 bp SeV GGATCACTAGGTGATATCGAGC* ACCAGACAAGAGTTTAAGAGATATGTATC* 181 bp *SeV SeV Transgene SeV IVQ&A Q1 A1 SeV SeV Q2 ips A2 ips Q3 ipssev A3 SeVSeV SeViPS SeV C5 11

12 V 1) Medical Science Digest 35(12): ips 2) Efficient induction of transgene free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Proc Jpn Acad Ser B Phys Biol Sci. 85(8): (2009) 3) ISBN ) 27(4): (2008) ESiPS 1 5) 57(1): (2007) ) 51(1): (2006) 7) ISBN C0345 (2006) 8) A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression. Li HO, Zhu YF, Asakawa M, Kuma H, Hirata T, Ueda Y, Lee YS, Fukumura M, Iida A, Kato A, Nagai Y, Hasegawa M. J Virol. 74(14): (2000) 12

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