ips CytoTune -ips ver.1.0 1
I CytoTune ips 3 II 3 3 4 5 III CytoTune ips ips 6 6 6 6 7 1) 2) ips 8 ips 8 SeV 9 SeV 10 IV Q&A 11 V 12 12 VI...13 13...13 2
I CytoTune ips CytoTune ips 4 OCT3/4SOX2 KLF4c MYCSeV ips SeV ips SeV ips II. SeV 1 RNA 2 naïve T 3 4 5 6 610 24 7 8 9 SeV RNA Okano, S. et al. Gene Ther, 10, 1381 1391(2003). 3
Sendai virus, SeV 1 SeV 1950 SeV HVJHemagglutinating Virus of Japan 1 RNA 15,384 150 250 nm 3 Nucleocapsid Protein, NPRNA Phosphoprotein, P Matrix protein, M (Fusion protein, F)/ Hemagglutinin Neuraminidase, HNRNA Large protein, L 6 4
SeV NPPMFHNL F SeV SeV F SeV/TS F SeV/TS15 F DNA SeV RNA SeV 3732204 3638019 3991339 3602058US 6,723,532US 6,645,760US 7,101,685US 7,442,544US 7,226,786US 6,746,860CN ZL96199467.3CN ZL96199476.2CN ZL805673.0 HK 1018287HK 1018078EP 0864645EP 1186667KR 552387KR 525687KR 10 0739938 CA 2236113 5
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 1x10 6 3 GenBank Accession No. OCT3/4NM_002701.4 SOX2: NM_003106.2 KLF4: BC029923.1 c MYC: K02276.1 80C 1 6
CO 2 (Φ100 mm 6 12 ) (15 ml) (5 ml10 ml) (200 µl1000 µl) IVF Cat. No. H 290 310, Cat. No. H 9570, (D MEM ) Basic fibroblast growth factorbfgf: human recombinant ES (4ng/mL bfgf ) ROCK :Y27632 0.1 % ( C MEF) ES ES 0.25% EDTA Fetal Bovine Serum FBS PBS anti Sendai Virus Cat. No. PD029, MBL 7
ips BJ ATCC No. CRL2522 ips ips 1. BJ 2 5x10 5 cells/well 6 2 2. CO 2 37C5% CO 12 3. 80 C CytoTune ips 37 C 4. 15 ml 10% FBS/D MEM 2 ml 4 ADOCT3/4SOX2KLF4c MYC 10 6 MOI=3 5 ml 5 5 5. 2 BJ 4 CytoTune ips 6. CO 2 37C5% CO 6 7. 24 10% FBS/D MEM 2 ml/well 8. CO 2 37C5% CO 56 10% FBS/D MEM 9. 10 * 11.5 10 6 cells/100mm dish MEF 10% FBS/D MEM *0.1% 4 ml/100mm dish 1 ml/well 612 well 37C 30 10. 67 PBS 1 PBS 5 0.25% EDTA 500 µl/well 10% FBS/D MEMiPS 8
11 11. 9 5 10 4 2 10 5 cells/100mm dish RT PCR SeV 12. CO 2 37C5% CO 13. 24 ES 37C3% CO 2 HEPES ES 5% 14. 15 6 1.72.510 5 cells/well 12 59 10 4 cells/well MEF 15. 20 14 6 IVF 10µM ROCK ES 16. CO 2 37 C3% CO 2 HEPES ES 5% 6 17. ES 18. 15 57 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
[] anti Sendai Virus 12 ips PBS 1 ml 10NWAKO 5 PBS 2 500 µl anti Sendai Virus 0.1% TritonX 100/ PBS 1/500 37 C 1 PBS 3 500 µl anti rabbit IgG 0.1% TritonX 100/ PBS 1/500 37 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 30 35 2% PCR 10
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 303839C5 11
V 1) Medical Science Digest 35(12): 505 5082009 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):348 362 (2009) 3) ISBN 97847581017452008 4) 27(4): 139 147(2008) ESiPS 1 5) 57(1): 29 36 (2007). 54 2. 6) 51(1): 27 37 (2006) 7) ISBN4 00 006274 3 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):6564 6569 (2000) 12
VI () P2 ips 13
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