Transcription of CytoTune™ - dnavec.co.jp
1 IPS .. CytoTune -iPS CytoTune .. 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 OCT3/4 SOX2 . KLF4 c MYC SeV . iPS . SeV iPS .. SeV .. iPS . II.. SeV 1 RNA . 2 . na ve T . 3 4 5 6 6 10 24 7 8 9 SeV RNA .. Okano, S. et al. Gene Ther, 10, 1381 1391(2003). 3.. Sendai virus, SeV . 1 SeV 1950 . SeV HVJ Hemagglutinating Virus of Japan . 1 RNA 15,384 150 250 nm . 3' Nucleocapsid Protein, NP RNA Phosphoprotein, P . Matrix protein, M . (Fusion protein, F) / . Hemagglutinin Neuraminidase, HN RNA . Large protein, L 6 .. 4.. SeV NP P M F HN L . F SeV . SeV F .. SeV/TS F SeV/TS15 F . DNA . SeV . RNA . SeV . 3732204 . 3638019 3991339 3602058 US 6,723,532 US 6,645,760 US 7,101,685 US 7,442,544 US 7,226,786 US 6,746,860 CN CN CN.
2 HK 1018287 HK 1018078 EP 0864645 EP 1186667 KR 552387 KR 525687 KR 10 0739938 . CA 2236113 5. III CytoTune iPS iPS 6. A OCT3/4 SeV/TS F 100 L 3 3 x 10 CIU / 100 L 6. B SOX2 SeV/TS F 100 L 3 3 x 10 CIU / 100 L 6. C KLF4 SeV/TS F 100 L 3 3 x 10 CIU / 100 L 6. D c MYC(HNL) SeV/TS15 F 100 L 3 3 x 10 CIU / 100 L 6. BJ 1x10 3 . GenBank Accession No.. OCT3/4 SOX2: KLF4: c MYC: . 80 C 1 6.. 1 CO2 ( 100 mm 6 12 ) (15 mL) (5 mL 10 mL) (200 L 1000 L) IVF Cat. No. H 290 310, Cat. No. H 9570, 2 . (D MEM ) Basic fibroblast growth factor bFGF : human recombinant ES (4ng/mL bFGF ) ROCK :Y27632 % ( C MEF) ES ES EDTA Fetal Bovine Serum FBS PBS anti Sendai Virus Cat. No. PD029, MBL 7. iPS BJ ATCC No. CRL2522 iPS . iPS . iPS 5. 1. BJ 2 5x10 cells/well 6 2 .. 2. CO2 37 C 5% CO 1 2 . 3. 80 C CytoTune iPS 37 C .. 4. 15 mL 10% FBS/D MEM 2 mL 4 . A D OCT3/4 SOX2 KLF4 c MYC . 6. 10 MOI=3 5 mL . 5 5 5.
3 2 BJ 4 . CytoTune iPS . 6. CO2 37 C 5% CO 6 7. 24 10% FBS/D MEM 2 mL/well .. 8. CO2 37 C 5% CO 5 6 10% FBS/D MEM . 9. 10 . 6. * 1 10 cells/100mm dish MEF . 10% FBS/D MEM * 4 mL/100mm dish 1 mL/well 6 12 well . 37 C 30 10. 6 7 PBS 1 PBS 5 . EDTA 500 L/well . 10% FBS/D MEM iPS .. 8. 11 4. 11. 9 5 10. 5. 2 10 cells/100mm dish RT PCR SeV . 12. CO2 37 C 5% CO 13. 24 ES 37 C 3% CO2 HEPES. ES 5% 14. 15 . 5. 6 10 cells/well 12 5 9. 4. 10 cells/well MEF 15. 20 . 14 6 IVF . 10 M ROCK ES 16. CO2 37 C 3% CO2 HEPES ES . 5% 6 17. ES 18. 15 5 7 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 30 38 39 C 3% CO2 HEPES . 5% 5 SeV . iPS V . 9. [ ] anti Sendai Virus 12 iPS PBS 1 mL 10N WAKO 5 PBS 2 500 L anti Sendai Virus TritonX 100/ PBS 1/500 37 C 1 . PBS 3 500 L anti rabbit IgG TritonX 100/ PBS 1/500 37 C. 1 PBS 3 Transgene SeV RT PCR iPS RNA 10.
4 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 product transgene Forward Reverse 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 IV Q&A Q1 A1 SeV .. SeV . Q2 iPS A2 iPS . Q3 iPS SeV A3 SeV SeV . SeV iPS . SeV . 30 38 39 C 5 . 11. V 1) Medical Science Digest 35(12): 505 508 2009 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.
5 85(8):348 362 (2009) 3) ISBN 9784758101745 2008 4) 27(4): 139 147(2008) ES iPS 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.. 300-2611. 6 . E-mail .. 460-0002. 3 5 10 5 . TEL: 052-971-2089 . E-mail 14.
