AAVpro® Helper Free System (AAV-U6
Contents
1. 0 5 ml AAV Extraction Solution A n 15 5 15 2 000 2 X 4C 10 50 ul AAV Extraction Solution 1 AAV 3 5 AAV 80 2 AAV Extraction Solution B ON VIII 1 AAV PCR2. TafdR4 Tel 077 565 6999 Fax 077 565 6995 http www takara bio coJp JND TARS v201508Da
3. pAAV 2xU6 Vector 6661 6662 6663 2 shRNA 2 shRNA hU6 mU6 2 1 2 shRNA 5 U6 A a K 5 RNA 3 UUUU J Dicer w siRNA UUUU http www takara bio coJp 4 6658 6663 pRC Vector AAV2 Rep Cap PRC2 mi342 Vector 2 2 6658 6661 pRC5 Vector 5 6659 6662 pRC6 Vector 6 6660 6663 pHelper Vector E2A E4 VA 2 AAV2 pRC Vector pRC2 mi342 Vector miRNA hsa miR 342
4. Adeno Associated Virus AAV AAV AAV 4 7kb 1 DNA ITR Inverted Terminal Repeat T 1 ITR AAV 3 ORF 1 Rep 2 Cap 3 AAP Rep 4 Rep78 Rep68 Rep52 Rep40 Cap 3
5. Lee er g 9 Paddison er g710 Paul er ag 11 Sui er g 12 1 2 shRNA 2 shRNA TTTTTT T 4 pol ll siRNA T 4 DNA Top strand T B 4 12 VII http www takara bio coJp 9 6658 6663 ARY LING 2 F amp Q x 91 ASAI ALO VNG E r pXISc0SC02 gt 6 2 38 q 2 VNG E NEEL r 21 AE 4 C LIHER 4 y Gesa pZ uanbas HMS X E DDDDLD9YYLLD YYDLLYY S 2 lt C HEMNE CYNA ODIO VNtuus E
6. AAV 16 1 P1 AAV jin vivo transduction ITR ITR VP1 VP3 AAP 1 AAV http www takara bio coJp 3 6658 6663 l 2 RNA RNA interference RNAi RNAi RNA
7. ea CA KA e AAV AAVpro Titration Kit for Real Time PCR Ver 2 6233 AAVpro Titration Kit for Real Time PCR Ver 2 PCR ITR ZsGreen1 AAV ZsGreen1 AAV pAAV ZsGreen1 6231 AAVpro Helper Free System 1 10 FBS DMEM 2 4 x 104 cells ml
8. 3 a CalPhos Mammalian Transfection Kit 631312 b 77ansIT 293 Transfection Reagent MIR2704 c Xfect Transfection Reagent 631317 a b AAV a CalPhos Mammalian Transfection Kit AAV CalPhos Mammalian Transfection Kit 2 X HEPES Buffered Saline 2 2 MCalcium Solution Sterile H20 6 333 mM Calcium Solution 3 DNA pAAV Vector 1 ug ul 6 ul pRC Vector 1 ug ul 6 ul pHelper Vector 1 ug ul 6 HI Calcium Solution 333 mM 1 000 ul Total 1 018 9 4 2 XHEPES Buffered Saline
9. 3 2 X HEPES Buffered Saline 15 5 3 3 3 DNA 6 HEK293 HEK293T 5 http wwwr takara bio coJp 13 6658 6663 b 7ransIT 293 Transfection Reagent 1 7ransIT 293 Transfection Reagent 2 DMEM DNA pAAV Vector pRC Vector pHelper Vector DMEM L lt Opti MEM Total SHI 3 2 45 ul O 7ransIT 293 Transfection Reagent 15 30 1 ug ul
10. l AAV 2 3 R J AAV http wwwr takara bio coJp 11 6658 6663 VII VII 1 In Fusion HD Cloning Kit pAAV U6 ZsGreen1 Vector pAAV 2xU6 Vector DNA pAAV U6 ZsGreen1 Vector 1 DNA 1 DNA Top strand Bottom strand 20 pmol ul DNA 2 95 5 30 25 2 pAAV U6 ZsGreen1 Vector Xbal Spel Stuffer sequence 900 bp 6 kb 3 In Fusion HD Cloning Kit In Fusion 5 X In Fusion HD Enzyme Premix 2 ul DNA 50 100 ng DNA 3 pmol dH2O Xul Total 10 ul 50
11. DNA 5 DNA http wwwr takara bio coJp 16 6658 6663 X 1 pRC2 mi342 Vector AAV2 O AAVpro Helper Free System pRC2 mi342 Vector AAV2 pRC2 mi342 Vector AAV2 HEK293 AAV2 AAV2 DpAAV CMV AcGFP1 Vector pRC2 mi342 Vector pRC2 Vector pHelper Vector 125
12. T 4 DNA Top strand T 4 lt Xba l pel gt Xba target sequence sense Hairpin loop target sequence antisense Terminator 5pel Top 5 pCTAGA G A NNNNNNNNNNNNNNNNNNN CTGTGAAGCCACAGATGGG NNNNNNNNNNNNNNNNNNN OCT TTTTTT A 3 Bottom 3 T C T NNNNNNNNNNNNNNNNNNN GACACTTCGGTGTCTACCC NNNNNNNNNNNNNNNNNNN G AAAAAAA TGATCp 5 lt Sal l Xho gt Sall target sequence sense Hairpin loop target sequence antisense Terminator Xhol Top 5 pTCGAC G A NNNNNNNNNNNNNNNNNNN CTGTGAAGCCACAGATGGG NNNNNNNNNNNNNNNNNNN OCT TTTTTT C 3 Bottom 3 G C T NNNNNNNNNNNNNNNNNNN GACACTTCGGTGTCTACCC NNNNNNNNNNNNNNNNNNN G AJAAAAAA GAGCTp 5 G A G A DNA
13. 5g Xho l DNA Top strand Bottom strand 2 N pol ll G A G A G A CTGTGAAGCCACAGATGGG Boden etal GTGTGCTGTCC Miyagishi efa 8 Lee er g79 Paddison er ga 10 Paul er g 1 Sui er g 12 1 2 shRNA 2 shRNA TTTTTT T 4 pollll siRNA
14. 2 24 well plate 0 5 ml well 3 AAV 10 FBS DMEM 1 000 100 000 4 2 Trypsin EDTA ZsGreen1 http wwwr takara bio coJp 15 6658 6663 X 2 DNA RNA DNA Ligation Ligation DNA pAAV U6 ZsGreen1 X a Spel pAAV 2xU6 Vector 2 1 Xba Spe 1
15. 6658 6663 I 3 A 2 II 1 2 AV AAVpro Helper Free System AAV2 U6 ZsGreen1 6658 pRC2 mi342 Vector 1 u g u pHelper Vector 1 u g ul AAV Extraction Solution A AAV Extraction Solution B Oi Paqo s SE DAAV U6 ZsGreen1 Vector 1 u g ul 20 ul 20 ul 20 ul 1 5 mlx 3 150 ulX 3 MM Helper Free System AAV2 2xU6 6661 DAAV 2xU6 Vector 1 u g ul pRC2 mi342 Vector 1 u g u pHelper Vector 1 u g ul AAV Extraction Solution A AAV Extraction Solution B TS OR 5 150 IX 3 AAVpro Helper Free System AAV5 U6 ZsGreen1 6659 pRC5 Vector 1 u g ul pHelper Vector 1 u g ul AAV Extraction Solution A AAV Extraction Solution B pAAV U6 ZsGreen1 Vector 1 u g ul 20 ul 20 ul 20 ul 1 5 ml x 3 150 ulX 3 Helper Free System AAV5 2xU6 6662 DAAV 2xU6 Vector 1 u g ul pRC5 Vector 1 u g ul pHelper Vector 1 ug ul AAV Extraction Solution A AAV Extraction Solution B Ce Oc 6 20 ul 20 ul 20 ul 1 5 ml x 3 150 ulX 3 AAVpro Helper Free System AAV
16. pRC2 Vector pRC mi342 Vector hsa miR 342 Vector AAV2 PCR 9 hsa miR 342 pRC2 mi342 Vector pRC2 Vector 2 AAV2 4 5 x 101 40 x 101 3 5 x 101 3 0 x 10 2 5 x 10 2 0 x 10 1 5 x 101 1 0 xX 1010 50 Xx 109 0 _3 86 x 1010 Total vg 1 89 x 1010 a a a oO O O O O O O O pRC2 pRC2 mi342 vg Vector Genome 9 AAV2 pRC2 mi342 http wwwr takara bio coJp 17 6658 6663 XI 2 AAV Extraction Solution c K Z AAV AAV Extraction Solution A B AAV AAVpro Extraction Solution 6235 XI 1 1 ZsGreen1 AAV
17. HEK293 AAV Extraction Solution AAV AAV PCR HT1080 10A B AV AAV 9 0 x 1010 7 0 x 1010 1 2 x 1012 10 NM 6 0 x 1010 1 0 x 1012 7 0 x 1010 E 0 x t 60x100 8 0 x 1011 50X1010 4 0 x 1010 Z 6 0 x 101 5 4 0 X 1010 3 0 x 1010 8 30x 1010 4 0 x 1011 S 20 x 1010 2 0 x 1010 10 x 1010 JS B 0 0 0 AAV2 AAV5 AAV6 I AES AAV Extraction Solution vg Vector Genome F T 10A AAV Extraction Solution AAV 35 30 25 20 15 AES AAV Extraction Solution 10 F T ZsGreen1 AES F T 10B AAV Extr
18. VP1 VP2 VP3 AAV 100 2 5 6 2 AAV2 1 5 AAV5 6 AAV6 AAV5 AAV6 L C XII AAV AAV AAV
19. 15 In Fusion HD Cloning Kit 4 co7HST08 Premium Competent Cells 9128 pAAV 2xU6 Vector pAAV 2xU6 Vector 2 pAAV U6 ZsGreen1 Vector DNA DNA pAAV 2xU6 Vector Xba 1 5pel 4 5 kb Sall Xho 4 9 kb VIl 2 VII 1 DNA DNA 1 ug ul NucleoBond Xtra Midi Max
20. hsa miR342 AAV2 miRNA miRNA Rep Cap pRC2 Vector 2 X 1 B AAV Extraction Solution AAV AAV AAV Extraction Solution AAV pAAV Vector pRC Vector pHelper Vector J AAV Extraction Solution O 39 2 AAVpro Helper Free System AAV QG AAV Ve http www takara bio coJp 5
21. Spel ITR Stuffer sequence Xbal Mouse U6 promoter ColE1 ori pAAV U6 ZsGreen1 Vector CMV 6 952 bp promoter Ampr Human B globin intron ITR hGH ZsGreen1 polyA 3 pAAV U6 ZsGreen1 Vector ColE1 ITR pAAV 2xU6 Vector 5 409 bp Spel Stuffer sequence1 Xbal T U6 promoter Human U6 promoter Sall Ampr Stuffer sequence2 Xhol 4 pAAV 2xU6 Vector ITR http www takara bio coJp 7 6658 6663 pRC2 mi342 Vector 8 189 bp HSV TK AAV2 Cap hsa miR 342 CMV promoter 5 pRC2 mi342 Vector Ampr pRC5 Vector 7 294 bp 6 pRC5 Vector ColE1 Ori AAV2 Rep Ampr pRC6 Vector 7 330 bp Cap 7 pRC6 Vector http www takara bio coJp 8 6658 6663 Ampr ColE1 ori Adenovirus E2A pHelper Vector Adenovirus 11 635 bp VA Adenovirus E4 8 pHelper Vector III 20 C AAV Extraction Solution A AAV Extraction Solution B 2 IV RNA
22. DNA RNA DNA In Fusion HD Cloning Kit 639648 RNA 15 15 DNA In Fusion DNA Top strand Bottom strand 2 N pol ll G A G A G A Xba l Spe shRNA oligo DNA 1 Sall Xhol shRNA oligo DNA 2 CTGTGAAGCCACAGATGGG Boden etal GTGTGCTGTCC Miyagishi er a 8
23. 1 0 5 M EDTA pH8 0 1 80 2 1 750 x g 10 P 55 S http wwwr takara bio coJp 14 6658 6663 VII 7 AAV AAV AAV Extraction A AAV AAV 2 1 VI 6
24. 5 ul 1 ug ul 5 ul 1 ug ul 5 ul 1 500 ul 1 515 ul HEK293 HEK293T 3 c Xfect Transfection Reagent 1 2 SO VII 5 Xfect Polymer Xfect Reaction Buffer DNA 5 pAAV Vector Tug ul 13 ul pRC2 Vector 1 ug ul 13 ul pHelper Vector 1 ug ul 13 ul Xfect Reaction Buffer 561 ul Total 600 ul 2 11 7 UIO Xfect Polymer 10 10 1 HEK293 HEK293T 6 2 FBS DMEM CalPhos Mammalian NN Kit VII 6 AAV 2
25. DDYDDDLY DLLYY L1D9YY S 2 lt C HEESNI amp nH LYNAI VNuus gt L ELEXA 022E NHR V PIAR 5 ARONEN lt Z E FISEN V FIAR 5 HERHOR A4 k S LDDLY YLDLDLD Y VVVVVV W5 NNNNNNNNNNNNNNNNNNN V1 19155 1 1 V VD NNNNNNNNNNNNNNNNNNN 1 1 9V 1 91 1 E puens woog VDDV1 1VDVDV LLLLLL UD NNNNNNNNNNNNNNNNNNN 59 1VDVOV 5VV 1 1 NNNNNNNNNNNNNNNNNNN V D V 19V5 vV VVV9 S puens dol KHAR JOleuluuel su snue guanbas abJe1 dool uidJleH 3suas aouanbas 1a5Je f KIHE xn S Bd 5 38 lt WB HS08Et ni S G Y ZX 2 EWE ELA Y 2 E H gt 4 4 4 1 ouX IIes VNO OBIIO VNJdU5 S LYLD YLYLDVDLY VVVVVV V D NNNNNNNNNNNNNNNNNNN V1 19195 11 V VD NNNNNNNNNNNNNNNNNNN 12 D 1V5V995D21111212 puells woog 4 V1VD 1V1VD1DV1 LLLLLL LD NNNNNNNNNNNNNNNNNNN 559 1V5V V 9VV 1 1 NNNNNNNNNNNNNNNNNNN WD 5V1 1 9VVVVDV9 s puens dol wP Hist JOJBUIUUJ91 su snue gouanbas ae1 dool ulduleH Ssuas aouanbas Ja5Je KIHE x wE lt ER HERE MGk ILG MRE EECA ea HRS 1 ds I eqx L VNG opllo YNYYS VN WS WY uolsnj ul 6658 6663 10 http wwwr takara bio
26. HEK293T 17 ATCC CRL 11268 AAV AAV AA pAAV ZsGreen1 Vector ZsGreen1 AAV lt AAV 6661 6662 6663 DNA 1 x PCR TaKaRa Ex Taq RR001A 10 X Ex Taq Buffer 20 mM Mg2 plus AAV DAAV U6 ZsGreen1 Vector pAAV 2xU6 Vector DNA 1 pAAV Vector HEK293 HEK293T pAAV Vector pRC Vector pHelper Vector HEK293 HEK293T
27. LacZ 6657 AAVpro Packaging Plasmid AAV2 6234 AAVpro Packaging Plasmid AAV5 6664 AAVpro Packaging Plasmid AAV6 6665 XV AAVpro TaKaRa Ex Taq In Fusion Clontech Laboratories Inc CalPhos Xfect Lenti X Clontech Laboratories Inc
28. mRNA 21 23 RNA short interfering RNA SIRNA RNAi RNAi siRNA siRNA siRNA RNAi RNAi AAVpro Helper Free System AAV U6 ZsGreen1 AAVpro Helper Free System AAV 2xU6 RNA polymerase III poll 2 5 6 AAV AAV Helper Free System AAV RNA shRNA AAV 1 2 AAV AAVpr
29. 6 U6 ZsGreen1 6660 pRC6 Vector 1 u g ul pHelper Vector 1 ug ul AAV Extraction Solution A AAV Extraction Solution B SR pAAV U6 ZsGreen1 Vector 1 u g ul 20 ul 20 ul 20 ul 1 5 ml 3 150 ulX 3 0 Helper Free System AAV6 2xU6 6663 DAAV 2xU6 Vector 1 u g ul pRC6 Vector 1 u g u pHelper Vector 1 u g ul AAV Extraction Solution A AAV Extraction Solution B lh OO http www takara bio co jp 20 ul 20 ul 20 ul 1 5 ml 3 150 ulX 3 6 6658 6663 Ready to use 2 3 AAVpro Packaging Plasmid AAV2 6234 pRC2 mi342 Vector 1 u g ul 0 5 ml x 2 pHelper Vector 1 ug ul 0 5 ml x 2 AAVpro Packaging Plasmid AAV5 6664 pRC5 Vector 1 u g ul 0 5 ml x 2 pHelper Vector 1 ug ul 0 5 ml Xx 2 AAVpro Packaging Plasmid AAV6 6665 pRC6 Vector 1 u g ul 0 5 ml x 2 pHelper Vector 1 ug ul 0 5 ml Xx 2 Il 2
30. Solution AAV SDS PAGE 100 90 80 70 60 50 40 30 7 77 10 0 j AAV Extraction Solution ug 1 X 1012 vg 12 AAV Extraction Solution AAV2 2 DNA http wwwr takara bio coJp 19 6658 6663 XII 3 shRNA shRNA AAV2 pAAV U6 ZsGreen1 1 2 1 0 0 8 1 0 6 Expression ratio NTD 0 4 0 2 0 AAV2 1 2 AAV2 shRNA XIII Miyake K et al 2012 J Nippon Med Sch 79 6 394 402 Van Vliet KM et al 2008 Methods Mol Biol 437 51 91 Wu Z et al 2006 Mol Ther 14 3 316 327 et al 2007 52 10 1288 1293 Zincarelli C et al 2008 Mol Ther 16 6 1073 1080 Ellis BL et al 2013 Virol J 10 74 7 Boden et al 2004 Nucleic Acids Res 32 1154 1158 8 Miyagishi et al 2004 J Gene Med 6 715 723 9 Lee et
31. action Solution AAV2 HT1080 http wwwr takara bio coJp 18 6658 6663 XI 2 2 AAV HEK293 AAV Extraction Solution AAV AAV PCR 1 X 10 vg SDS PAGE 11 AAV2 2 DNA 2 DNA 12 AAV Extraction Solution 2 DNA AAV2 AAV5 AAV6 PT AES F T AES F T AES 3 F T AES AAV Extraction Solution 1 x 109 vo lane ll 11 AAV Extraction
32. al 2002 Nat Biotech 20 500 505 10 Paddison er al 2002 Genes and Dev 16 948 958 11 Paul et al 2002 Nat Biotech 20 505 508 12 Sui et al 2002 Proc Natl Acad Sci USA 99 5515 5520 Q N http wwwr takara bio coJp 20 6658 6663 XIV In Fusion HD Cloning Kit 639648 CalPhos Mammalian Transfection Kit 631312 TransIT 293 Transfection Reagent MIR2704 MIR2700 MIR2705 MIR2706 Xfect Transfection Reagent 631317 631318 Lenti X 293T Cell Line 632180 PAAV ZsGreen1 Vector 6231 AAVpro Purification Kit AAV2 6232 AAVpro Titration Kit for Real Time PCR Ver 2 6233 AAVpro Extraction Solution 6235 AAVpro Helper Free System AAV2 6230 AAVpro Helper Free System AAV5 6650 AAVpro Helper Free System AAV6 6651 AAVpro Helper Free System AAV2 CRE Recombinase 6652 AAVpro Helper Free System AAV5 CRE Recombinase 6653 AAVpro Helper Free System AAV6 CRE Recombinase 6654 AAVpro Helper Free System AAV2 LacZ 6655 AAVpro Helper Free System AAV5 LacZ 6656 AAVpro Helper Free System AAV6
33. coJp V V 1 100 mm VI V 2 In Fusion HD Cloning Kit 639648 a CalPhos Mammalian Transfection Kit 631312 b TransIT 293 Transfection Reagent MIR2704 c Xfect Transfection Reagent 631317 Dulbecco s Modified Eagle s Medium DMEM 4 5 g L Glucose with L Glutamine 584 mg L Fatal Bovine Serum FBS Trypsin EDTA HEK293 HEK293T 1 0 5 M EDTA pH8 0 EDTA Buffer Powder pH8 0 T9191 PpAAV ZsGreen1 Vector 2 6231 DNA 10 mM Tris HCI pH8 0 50 mM NaCl 3 x1 x2 x3 HEK293 HEK293T AAV Lenti X 293T Cell Line 632180
34. i 740410 10 740414 10 DNA 13 500 x 9 5 DNA http wwwr takara bio coJp 12 6658 6663 VII 3 HEK293 HEK293T HEK293 HEK293T 100 mm 2 5 40 X 106 cells dish 10 FBS DMEM VII 4 CalPhos Mammalian Transfection Kit Xfect Transfection Reagent 10 ml L TranslT 293 Transfection Reagent 15 ml VII 4 pAAV Vector pRC Vector pHelper Vector HEK293 HEK293T VII 2 pAAV Vector pRC Vector pHelper Vector VI 3
35. o Purification Kit AAV2 6232 AAV A AAV Helper Free System AAV AAV Helper Free System AAV 2 AAV HEK293 AAV 3 AAVpro Helper Free System 6 shRNA pAAV Vector shRNA 2 ITR pAAV U6 ZsGreen1 Vector 6658 6659 6660 1 shRNA mU6 shRNA CMV ZsGreen1 ZsGreen1
36. wa r 6658 6663 IdfaR4 AAVpro Helper Free System AAV U6 ZsGreen1 AAV 2xU6 vV201508Da 16 1 P1 AM DNA http wwwr takara bio coJp 2 6658 6663 l l 1
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