GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. Premade Lentiviral Particles for iPS Stem Factors human set For generating induced pluripotent stem iPS cells or other applications RESEARCH USE ONLY not for use in diagnostics or therapeutics Product Name h OCT4 RFP Bsd inducible particles 200ul x LVP003 1 x108 IFU ml o h SOX2 RFP Bsd inducible particles 200ul x LVP004 1 x108 IFU ml h NANOG RFP Bsd inducible particles 200ul x LVP005 1 x108 IFU ml h LIN28 RFP Bsd inducible particles 200ul x LVP006 1 x108 IFU ml h cMyc RFP Bsdq inducible particles 200ul x LVP007 1 x108 IFU ml ioe h Kif4 RFP Bsd inducible particles 200ul x LVP008 1 x10 IFU ml amen Full set 6 human stem factors with RFP emma LVP stems h Bsd marker 200ul ea x 6 Storage lt 70 C avoid repeat freeze thaw cycles Products stable for 6 month Product Description The lentiviral system is a gene delivery tool using lentivectors for gene expression or knockdown Lentivectors are HIV 1 Human Immunodeficiency Virus 1 derived plasmids and are used to generate lentiviral particles lentivirus that can be transduced virtually all kinds of mammalian cell types or organs including stem cells primary cells and non dividing cells both in vivo and in cell culture system Particles stably integrate into the transduced cells genome for long term expression Therefore lentivirus holds a unique promise as a gene transfer agent Converting fully differentiated2. human or mouse somatic cells into embryonic like cells so called induced Pluripotent Stem Cell 1PSC has attracted enormous attention in stem cell research Multiple reports have demonstrated that iPS cells were generated by using a set of transcription factors or stem cell factors that can be delivered as expression virus or expressed proteins Although the combination of reprogramming factors may vary slightly the main stem cell factors are OCT3 4 SOX2 NANOG LIN28 cMyc and KLF4 Pre made Lentiviral Particles for iPS product manual Page 1 of 6 Switzerland Deutschland UK amp Rest of World amsbio Fax 49 0 69 13376880 into amsbio com Tel 49 0 69 779099 Centro Nord Sud 2E CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton Park Abingdon OX14 4SE UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 AMS Biotechnology Europe Ltd Registered in England amp Wales No 2117791 AMSBIO s pre made lentivirial particles for iPSC are generated from its proprietary lentiviral vector system with either RFP Bsd fusion dual marker or Neomycin marker No RFP see vector map scheme below Six human stem cell factors were first individually cloned into lentivector Then lentivectors were co transfected with a packaging mix Cat HT pack into a 293T packaging cells cat TLV C The pre made lentiviral particles are VS V G pseudotype
3. 0 split the transduced live cells into feeder cells using your defined medium continue to incubate for 24 48hrs Change to hES medium continue to incubate and change the hES medium every day 8 At days 13 18 you will observe the cells morphology changing and ES like colonies forming see sample image below IPSC generating from human fibroblast cells RFP filter 495nm 620nm Bright light Pre made Lentiviral Particles for iPS product manual Page 4 of 6 Switzerland Deutschland UK amp Rest of World amsbio TP a AL E AA nt a intToOwvamspio com Centro Nord Sud 2E Tel 49 0 69 779099 CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton Park Abingdon OX14 4SE UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 AMS Biotechnology Europe Ltd Registered in England amp Wales No 2117791 iPS cell generated by AMSBIO s iPS lentiviral particles Human Fibroblasts iPS clone at Day 14 after virus transfection Bright light image Live staining for TRA 1 60 iPS clone at Day 18 after virus transfection Safety Precaution Please use extra caution when using lentiviral particles Remember Wear gloves at all the times when handling Lentiviral particles Please note that although our lentiviral vectors contain all necessary bio safety features work with lentiviruses should be carried out under Biological Safet
4. d virus packaged in serum free medium and supplied as 200ul per vial at 1x 10 IFU ml schematic representation of inducible lentivector sy FFP Be E or Neo Stem Factors Oct3 SOX2 NANOG LIN28 c Myc or KLF4 Each stem factor was natively expressed without any tags under a tetracycline inducible suCMV promoter in which two tetracycline operator sequences was integrated For inducible expression the tetR must be expressed in advance to stop the transcription and expression is then activated by adding tetracycline This inducible expression is dose dependent In general the amount of tetracycline used is lug ml final concentration Please see the schematic below for the mechanism of inducible expression and see our website for more details about Inducible lentiviral system For particles general information please refer to FAQ about premade lentiviral particles All six stem factor were sequencing verified Their sequences fully match the CD region according to the NCBI s database see table below Lentiviral particles contain blasticidin RFP fusion marker which allows selection of the transduced cells by either fluorescence sorting or antibiotic selection Pre made Lentiviral Particles for iPS product manual Page 2 of 6 Switzerland Deutschland UK amp Rest of World amsbio Fax 49 0 69 13376880 into amsbio com Centro Nord Sud 2E Tel 49 0 69 779099 CH 6934 Bioggio L
5. dottir G A Ruotti V Stewart R Slukvin I I and Thomson J A 2007 Induced pluripotent stem cell lines derived from human somatic cells Science 318 1917 1920 5 Park I H et al Reprogramming of human somatic cells to pluripotency with defined factors Nature 2008 451 7175 p 141 6 6 Shao L et al Generation of iPS cells using defined factors linked via the self cleaving 2A sequences in a single open reading frame Cell Res 2009 19 3 p 296 306 7 NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors Link 8 CDC guidelines for Lab Biosafety levels Link Pre made Lentiviral Particles for iPS product manual Page 6 of 6 Switzerland Deutschland UK amp Rest of World Centro Nord Sud 2E Tel 49 0 69 779099 CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton Park Abingdon OX14 4SE UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 amsbio Fax 49 0 6 13376880 Tatto Ce emenelaa f IEN wt EE TS Be Ee Ne NFI EE AMS Biotechnology Europe Ltd Registered in England amp Wales No 2117791
6. r iPS product manual Page 3 of 6 Switzerland Deutschland UK amp Rest of World amsbio Fax 49 0 69 13376880 into amsbio com Centro Nord Sud 2E Tel 49 0 69 779099 CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton Park Abingdon OX14 4SE UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 AMS Biotechnology Europe Ltd Registered in England amp Wales No 2117791 Optional to check the transduction ability of your cells add 50ul of GFP His Control Lentiviral particles LVPOO2 into cells Check the expression level by GFP signal and transduction efficiency by RFP signal under a fluorescent microscope 3 Change to complete medium at 12hrs or at a longer time point after transduction please aware that longer transduction times can result in more cell death when KIf4 SOX2 and cMyc are over expressed and incubated again overnight Note use the serum free particles without any worry of unwanted differentiation 4 Check cell viability you may observe some cells dying floating up From now on change the medium every two days serum free medium 5 Check transduction efficiency at 72 hours by vitalizing the RFP signal under a fluorescent microscope using a RFP filter filer Ex 550 Em620nm You may observe that the majority of cells are dead but there will be some living cells showing a good RFP signal 6 At days 8 1
7. ugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton Park Abingdon OX14 4SE UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 AMS Biotechnology Europe Ltd Registered in England amp Wales No 2117791 suCMV amp zu 2o EE l E l Tet operator gene tetR protein tet Expression particles Expression repressed 1 tetR protein binds to tet operator sequences in promoter to repress the transcription 4 Add tet B Expression induced Tetracycline binds to tetR to release tetR from promoter and transcription is initiated hMyc NM_002467 hKIf4 NM_004235 h Oct3 4__ NM_002701 hSOX2 _ NM_003106 hLIN28 _ NM_024674 h NANOG NM_024865 NCBIID Matched ORF position Experimental Protocols for generating iPS cells for your reference only Note for the purpose other than generating iPS cells please follow our web link general transduction protocols under premade lentiviral particles 1 Seed the desired parent cells at 1x10 cells well in a 24 well plate and incubated overnight 2 Add 50ul of each lentivirial particle for iPSC Oct3 4 Sox2 NANOG LIN28 c Myc and KIf4 Note Polybrene has been reported to enhance virus transduction at 6 8ug ml final concentration But Polybrene could be toxic to some cell types so add as desired Notes You can set up your own factor combinations dependent upon your cell type Pre made Lentiviral Particles fo
8. y Level 2 BL 2 or higher Please conduct a thorough risk assessment for your project and contact your health and safety facilities for local guidelines and regulations Please refer CDC and NIH s links see references for more details regarding safety issues Pre made Lentiviral Particles for iPS product manual Page 5 of 6 Switzerland Deutschland UK amp Rest of World Centro Nord Sud 2E Tel 49 0 69 779099 CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton Park Abingdon OX14 4SE UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 amsbio Fax 49 0 69 13376880 Tatto Ce JolloWelolna AMS Biotechnology Europe Ltd Registered in England amp Wales No 2117791 Related Product Premade lentiviral particle for six mouse stem factors Cat SC015 h Oct3 4 stable cells 2 x 10 cells SC016 h LIN28 stable cells 2x 10 cells SCO17 h NANOG stable cells 2 x 10 cells References 1 NIH stem cell training program Link 2 Masaki Ieda Ji Dong Fu et al 2010 Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors Cell 142 375 386 3 Takahashi K and Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126 663 676 4 Yu J Vodyanik M A Smuga Otto K Antosiewicz Bourget J Frane J L Tian S Nie J Jons
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