pLVSIN Vector シリーズ
Contents
1. iei http www lifescience mext e e TA http www takara bio co jp 6 6181 6192 A Lenti X 293T Cell Line 632180 HEK 293T Contechniques October 2008 Lentiviral High Titer Packaging Mix pLVSIN 7g sIT 293 Transfection Reagent Lenti X 293T2. 4 ug9 m MOD 13 8 24 37 5 CO02 32 1 200 X g 60 90 32 37C 5 COo
3. Retroviruses ed by J M Coffin S H Hughes amp H E Varmus 1997 Cold Spring Harbor Laboratory Press NY Culture of Animal Cells 5th Edition ed by R I Freshney 2005 Wiley Liss NY Current Protocols in Molecular Biology ed by F M Ausubel et al 1995 Wiley amp Sons B Lenti X 293T A HEK 293
4. 80 1 2 1 4 HT 1080 MOI
5. HEK 293T American Type Culture Collection ATCC No CRL 121 HEK 293T 17 HEK293T HT 1080 cell line American Type Culture Collection HT 1080 ATCC No CCL 121 B 1 a TransIT 293 Transfection Reagent MIR2704 b CalPhos Mammalian Transfection Kit 631312 SIN 6187 6190 1 a b
6. Rs f fce us 48 C 1 48 72 5 LTR LTR 2 lt 9 7 kb 104 IFU ml CENTER neue pum SUED x 3 4 ug ml 2 12 ug ml mE Um cin EREDETU I 4 IVA TIS 1 4 1 2 ANT R z Ol JEZ Bitu antibiot
7. DNA RetroNectin In Fusion Clontech Laboratories Inc SYBR Life Technologies Corporation Lenti X CalPhos GoStix Clontech Laboratories Inc
8. Lentiviral High Titer O Packaging Mix 1 Lentiviral High Titer Packaging Mix Q pLVSIN Vector Lenti X 293T Packaging Cell OP 2 ZNN JI 9 7 88 3 T l SNIN A V RNA 4 ww 5 VSV G 0 6 1 Lentiviral High Titer Packaging Mix Lenti X 293T Lentiviral High Titer Packaging Mix pLVSIN 1 RNA 2 RNA V C
9. RetroNectin J RetroNectin RetroNectin Recombinant Human Fibronectin Fragment T100A B CH 296 Clontchniques October 2008 RetroNectin substratum RetroNectin http wwwr takara bio coJp 8 6181 6192 HI
10. http wwwr takara bio coJp 3 6181 6192 PLVSIN Vector Y AcGFP1 N C 6187 6190 ZsGreen1 2 IRES IRES 6191 6192 RES 1 AcGFP1 4eggo rea coerulescens 2 ZsGreen1 Zoanthus sp 1 AcGFP1 ZsGreen 1 nm 475 493
11. qRT PCR Lenti X qRT PCR Titration Kit 631235 1 qRT PCR SYBR Green RNA 4 p24 ELISA Lenti X p24 Rapid Titer Kit 632200 ELISA p24 p24 4
12. TransIT 293 Transfection Reagent 2 AcGFP1 C Lenti X 293T Cell Line 632180 HT 1080 cell line 45 g L Dulbecco s Modified Eagle s Medium DMEM 1096 FBS Penicillin Streptomycin solution 10 000 units ml Penicillin G sodium salt 10 000 ug ml Streptomycin sulfate Trypsin EDTA Dulbecco s phosphate buffered saline DPBS D 100 mm 12 1 5 ml 2 0 ml 15 ml
13. Appendix H LVSIN G418 631307 631308 Hygromycin B 631309 Puromycin 631305 631306 Appendix L Polybrene hexadimethrine bromide Sigma Aldrich No H9268 2 12 ug ml
14. TIafd4R4 Tel 077 565 6999 Fax 077 565 6995 http www takara bio co jp JND TARS v201509Da
15. HT 1080 HeLa 2 12 ugm 24 RetroNectin Recombinant Human Fibronectin Fragment T100A B VII 2 T 2
16. Lentiviral High Titer Packaging Mix Lenti X 293T Cell Line Lentiviral High Titer Packaging Mix VSV G VSV G CMV EF1a
17. 6 8 24 48 http wwwr takara bio coJp 15 6181 6192 VII 2 RBV spin A RetroNectin RetroNectin PBS Q0 100 ug mD 1
18. Human immunodeficiency virus HIV 1 pLVSIN 2 Lentiviral High Titer Packaging Mix P2 Lentiviral High Titer Packaging Mix 3 RCL replication competent lentivirus 3
19. Lenti X 293T Lentiviral High Titer Packaging Mix Lentiviral High Titer Packaging Mix Lenti X293T 1 2 DNA 3 pLVSIN Lentiviral High Titer Packaging Mix Lenti X293T HIV 1 V 1 Lenti X 293T Le
20. RetroNectin RetroNectin Bound Virus Spin RetroNectin RetroNectin coated plate RetroNectin Bound Virus Spin VII 2 D 1 6181 6192 IX Lentiviral High Titer Packaging Mix 6194 Lenti X 293T Cell Line 632180 Lenti X qRT PCR Titration Kit 631235 Lenti X p24 Rapid Titer Kit
21. A Coffin et al 1997 DNA NucleoBond Xtra Midi Maxi 740410 10 740414 10 DNA 14000 X g 10 DNA DNA http wwwr takara bio coJp 9 6181 6192 IV A
22. 1 10 1 100 PBS 10 100 http wwwr takara bio coJp 22 6181 6192 Xl 16 1 P2 4
23. 5 37 CO CO 5 1096 24 6 Ec ids 24 HEK 293 48 7 amp Ud 25 Lenti X 293T Cell Line Protocol at a Glance PT4058 2 http wwwr takara bio coJp 10 6181 6192 V pLVSIN
24. 1 25 ml 37 1 C 2 37C 70 1 ml 15 ml 3 4 ml 4 5 ml 100 x Xg 5
25. p24 Lenti X GoStix 631243 631244 20 ul 30 P 10 p24 F Lenti X Maxi Purification Kit 631233 631234 G Lenti X Concentrator 631231 631232 100 MO
26. a TranslT 293 Transfection Reagent MIR2704 TransIT 293 Transfection Reagent 1 TranslT 293 Transfection Reagent 2 2 0 ml DMEM DNA Lentiviral High Titer Packaging Mix 7 ul 0 5 ug ulpLVSIN Vector 11 ul DMEM 1 500 ul Total 1 518 ul http wwwr takara bio coJp 11 6181 6192 3 2 45 ul TransIT 293 Transfection Reagent 15 30 4 Lenti X 293T 3 5 CO02 37 b CalPhos Mammalian Transfection Kit 631312 CalPhos Mammalian Transfectio
27. B Lenti X GoStix 20 ul Chase Buffer GoStix Test Band 30 10 http wwwr takara bio coJp 13 6181 6192 B AcGFP1
28. AcGFP1 AcGFP1 MCS N ATG C1 Vector C1 MCS AcGFP1 MCS AcGFP1 C AcGFP1 AcGFP1 MCS AcGFP1 ATG MCS C C
29. mRNA cPPT CTS cPPT CTS DNA cPPT CTS Zennou etal 2000 RRE Rev RNA Cochrane et al 1990 3x pLVSIN Vector 6181 76186 EKU 6187 6190 AcGFP1 Neor Hyg Puro 6191 6192 ZsGreen1 http wwwr takara bio coJp 4 6181 6192 C Lentiviral High Titer Packaging Mix
30. 3 500 x g 1 37C 5 CO http wwwr takara bio coJp 16 6181 6192 VII 1 B Lenti X 293T 1 2 3 4 DNA DNAO B e 558074 e 5 i coli HST08 Premium Electro Cells Oo 3 LTR 9028 IV B ILC
31. B 3 1 1 ml 1096 FBS DMEM 2 5 CO2 37 B 4 3 Trypsin EDTA AcGFP1 B 5 Viral biologicaltiter AcGFP1 IFU ml AcGFP1 1 0 20 096 Titer IFU ml x AcGFP1 100 0 5 ml HT 1080 HT 1080 http wwwr takara bio coJp 14 6181 6192 VII fl VII 1
32. 0 45 um PolyVinylidene DiFluoride PVDF polyethersulfone PES http wwwr takara bio coJp 7 6181 6192 E SEU x d Lenti X qRT PCR Titration Kit 631235 qRT PCR Clontechniques January 2008 Quinn et al 1997 SYBR Green PCR RNA NNA 4 Lenti X p24 Rapid Titer Kit 632200 ELISA p24
33. MCS C C MCS IRES Vector MCS IRES ZsGreen1 mRNA ATG IRES MCS 0 7 1 2 kb IRES MCS ORF Kozak consensus ribosome binding site Kozak 1987 cDNA A LTR
34. RetroNectin 0 25 0 5 ml cm2 RetroNectin 2 2 4 24 1 0 5 ml 6 1 2 ml RetroNectin 2 RetroNectin 2 BSA PBS 30 3 24 0 5 m well 6 2 ml well 3 2 BSA PBS 4 PBS HBSS HEPES 2 2 BSA PBS PBS HBSS HEPES
35. Gr Ed zai 2c 1 ILC Phi pe pem 1 Lenti X 293T http wwwr takara bio coJp 17 6181 6192 C TransIT 293 Transfection Reagent amp 5X 106 cells 100 mm V Lenti X 293T PO TIR A 6o 280 1 8 2 0 DNA NucleoBond Xtra MARY o IMidi Maxi EF 740420 10 740424 10
36. 6183 pLVSIN EF1 a Neo Vector 6184 pLVSIN EF1 a Hyg Vector 6185 pLVSIN EF1 a Pur Vector 6186 SIN AcGFP1 pLVSIN AcGFP1 N1 Vector 6187 pLVSIN AcGFP1 CT Vector 6188 pLVSIN EF1 a AcGFP1 N1 Vector 6189 pLVSIN EF1 a AcGFP1 C1 Vector 6190 ZsGreen1 IRES PLVSIN IRES ZsGreen1 Vector 6191 PLVSIN EF1 a IRES ZsGreen1 Vector 6192 Lentiviral High Titer Packaging Mix 6194 Lenti X 293T Cell Line 632180 A SIN Self Inactivating pLVSIN Lentiviral High Titer Packaging Mix
37. 196 Penicillin Streptomycin DMEM CalPhos Mammalian Transfection Kit V 4 1 48 2 0 45 um 80 Higashikawa et al 2001 http wwwr takara bio coJp 12 6181 6192 VI A MOI
38. 632200 Lenti X GoStix 631243 631244 631241 Lenti X Maxi Purification Kit 631233 631234 Lenti X Concentrator 631231 631232 RetroNectin Recombinant Human Fibronectin Fragment T100A B RetroNectin Dish RetroNectin Pre coated Dish 35 mmo T110A http wwwr takara bio coJp 20 6181 6192 X Cochrane A W Chen C H and Rosen C A 1990 Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA Proc Natl Acad Sci USA 87 1198 1202 Coffin J M Hughes S H and Varmus H E eds 1997 Retroviruses Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Higashikawa F and Chang L 2001 Kinetic Analysis of stability of simple and complex retroviral vectors Virology 280 124 131 Higashimoto T Urbinati F Perumbeti A Jiang G Zarzuela A Chang L J Kohn D B and Malik P 2007 The woodchuck hepatitis virus post transcriptional regulatory element reduces readthrough transcription from retroviral vectors Gene Ther 14 17 1298 1304 Improve Viral Transductions with RetroNe
39. nm 505 505 BEY 26 650 39 130 cm 1M X B pLVSIN pLVSIN Vector SIN HIV 1 LTR 5LTR 3 LTR A U3 P WPRE WPRE A RNA RNA Zufferey et al 1999 Higashimoto et al 2007 WPRE
40. pLVSIN DNA coli HST08 Premium Electro Cells 9028 pLVSIN Vector In Fusion HD Cloning Kit 639648 PCR 1 3 1 2 Multi Cloning Site MCS CDNA SIN Vecor CDNA ATG N1 Vecor N1 MCS AcGFP1 MCS AcGFP1 N
41. RetroNectin 4 4 2 BSA PBS 4 1 B RetroNectin Bound Virus RBV Spin RetroNectin 125 250 ul cm 32 2 000 X 9 2 RetroNectin PBS 0 1 2 BSA HSA PBS 0 05 5 x 10 cells cm2
42. pLVSIN AcGFP1 N1 Vector 6187 Lentiviral High Titer Packaging Mix 7ranslT 293 Transfection Reagent B 1 HT 1080 HT 1080 12 2 5 X 104 cells 1 mwell 5 CO2 37 10 FBS DMEM 1 Penicillin Streptomycin DMEM B 2 1 1096 FBS DMEM 450 8 mg ml 0 5 ul 2 B 1 HT 1080 450 ul 3 10 FBS DMEM 20 2 000 4 50 2 HT 1080 8 ug ml 200 20 000 5 5 CO2 37
43. 3 ml 6 2 x 105 Puromycin 0 1 0 2 5 5 0 7 5 10 0 ug ml 2 G418 Hygromycin B 5 10 2 4 3 Puromycin 4 7 2 2 Lenti X Concentrator 631231 Lenti X Concentrator 30 ml DHA Concentrator 1 3 10 ml 4 30 O N 1 500 X g 4 45
44. Human immunodeficiency virus 1 HIV 1 16 1 16 7 HIV 1 2 3 1 HIV 1 nef vif vpr vou tat rev 2 gag pol env 3 LTR 3TLTR U3 HIV 1 17 10 14
45. Lentiviral High Titer Packaging Mix pLVSIN Packaging Mix Lenti X 293T Packaging Mix HIV 1 Gag Pol Tat Rev VSV G pLVSIN RNA 1 Packaging Mix Lenti X 293T Trans T 293 Transfection Reagent MIR2700
46. AT Y 3 RNA 4 VSV G 5 6 http www takara bio co jp 5 6181 6192 D pLVSIN
47. ctin Reagent October 2008 Clontechniques 2008 11 12 Kozak M 1987 At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells J Mol Biol 196 947 950 Lenti X 293T Cells for Superior Lentivirus Packaging October 2008 Clontechniques 2008 10 Quinn T P and Trevor K T 1997 Rapid quantitation of recombinant retrovirus produced by packaging cell clones Biotechniques 23 1038 1044 Rapid Lentiviral and Retroviral Titration Kits January 2008 Clontechniques 2008 19 21 Zennou V Petit C Guetard D Nerhbass U Montagnier L and Charneau P 2000 HIV 1 genome nuclear import is mediated by a central DNA flap Ce 101 173 185 Zufferey R Donello Trono D and Hope T J 1999 Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors J Virol 73 2886 2892 Coffin J M Hughes S H and Varmus H E eds 1997 Retroviruses Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Freshney R I 2005 Culture of Animal Cells 5th Edition Wiley Liss NY Ausubel F M Brent R Kingdom R E Moore D M Seidman J G Smith J A and Struhl K eds 1995 Current Protocols in Molecular Biology John Wiley amp Sons NY http wwwr takara bio coJ
48. es 9 IV A BMB 7 REC D PU WO A OBSS upset UR EON Ru 10 B Lenti X 2937 ftl EB tentent ttes tette ttt 10 V pLVSIN Lenti X 293T Lentiviral High Titer Packaging Mix 11 VI A 58 98 7 RR 79 725 ete ei eee d Ru RR EE be e en B VII 15 MI oi 17 Pp EN 20 NAM AU mit mec 21 Appendix 1 ee 22 SR RS 22 KE ME ES eis 23 http www takara bio co jp 2 6181 6192 Il SIN pLVSIN CMV Neo Vector 6181 pLVSIN CMV Hyg Vector 6182 pLVSIN CMV Pur Vector
49. ic kill curve http wwwr takara bio coJp 18 6181 6192 D 1 MOI 2 3 http wwwr takara bio coJp 19 C 2 Appendix
50. n Kit 1 2 X HEPES Buffered Saline 2 M Calcium Solution Sterile H O 2 DNA 15 ml Lentiviral High Titer Packaging Mix 7 ul 0 5 ug ulpLVSIN Vector 11 ul 2M Calcium Solution 87 ul Sterile H20 595 ul Total 700 ul 3 2 X HEPES Buffered Saline 2 700 ul 2 X HEPES Buffered Saline 15 4 5 5 DNA 5 Lenti X 293T 4 5 CO2 37 V 3 24 10 FBS DMEM 10 ml
51. nti X 293T 100 mm 5 0 Xx 108 cells 10 ml dish f amp L 5 CO2 37 1096 FBS DMEM 1 Penicillin Streptomycin DMEM 1 CalPhos Mammalian Transfection Kit 631312 2 5 X 106 cells 10 mdish V 2 pLVSIN Lentiviral High Titer Packaging Mix Lenti X 293T 2 a TransIT 293 Transfection Reagent MIR2704 b CalPhos Mammalian Transfection Kit 631312 a b 7ransIT 293 Transfection Reagent bET
52. p 21 6181 6192 Appendix 1 LVSIN G418 631308 Hygromycin B 631309 Puromycin 631306 1 Eo Sii ai HEMA OO O G418 50 800 ERE zm Hyoromdn 39 8 xo ioo 0252 CEST G418 Hygromycin B 5 2 Puromycin 3 4 1 G418 Hygromycin B 0 50 100 200 400 800 ug ml
53. usz r 6181 6192 Talana pLVSIN Vector pLVSIN Vector Lentiviral High Titer Packaging Mix 6194 v201509Da l A aese 3 B pLVSIN C Lentiviral High Titer Packaging MK DINALA CSATA WOULD edNN 6 ll A ee 7 Slot NAT 7 DU 7 pe i e lp 7 E DERE ATTORE srt tert itt edet RR these t P reete etre tesi dte 8 E dE or Ko D SUO 1 Bee cer pete OO on Repente cere e to Rad 8 GE DR A n 8 H 2s inpet tit etie c 8 8 J RetroNectin 8 Ill pLVSIN Le
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