Minicircle DNA and mc-iPS Cells Cat. #SC301A-1, SRMXXXPA-1
Contents
1. Sf MC LGNSO Minicircle Reprogramer D Catalog SRM100PA 1 Create Nonviral IPS Cells Phase SSEA4 NANOG 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual B Minicircle derived iPS cell line In addition to the pre made ready to transfect 4 in 1 minicircle reprogramming DNA SBI also offers the Human mc iPS Cell line highlighted in Nature Methods A nonviral minicircle vector for deriving human iPS cells Jia F et al 2010 Mar 7 3 197 9 The mc iPS cell line was derived from adult human adipose stem cells hASCs and is certified positive for pluripotency protein marker immunostaining and by gene expression Oct4 endo Sox2 endo Nanog endo Lin28 endo Page 4 ver 2 111910 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 The mc iPSCs also demonstrate multiple lineage potential Embryoid Bodies from mc iPSCs Iv Undifferentiated U Differentiated D Endothelial Cells Neurons Cardiomyocytes 5 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Protocols A Transfection of Minicircle DNA for reprogramming The following protocol has been optimized for human adipocyte derived stem cells according to the method described in Jia et al Other source cells may require transfection optimization for efficient reprogramming In general reprogramming requires approx2. in one well of a 6 well plate with MEF feeder cells Incubate the plate overnight at 37 C Change the medium every day until the cells reach 80 confluency If desired Y 27632 can be added into the culture media for a few days after thawing Usually it takes about one week to observe the iPSC colonies Page 8 ver 2 111910 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 lll References Fangjun Jia et al A nonviral minicircle vector for deriving human iPS cells Nature Methods 2010 Mar 7 3 197 9 Elayne Chan et al Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells Nature Biotechnology 27 11 1033 1037 November 2009 Zhi Ying Chen et al Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo Human Gene Therapy 16 1 126 131 January 2005 Zhi Ying Chen et al Minicircle DNA Vectors Devoid of Bacterial DNA Result in Persistent and High Level Transgene Expression in Vivo Molecular Therapy 8 3 495 500 September 2003 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual IV Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at
3. Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com System Biosciences SBI 1616 North Shoreline Blvd Mountain View CA 94043 V Licensing and Warranty Use of the mc iPS cell line and mc LGNSO DNA i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use Page 10 ver 2 111910 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research This Product shall be used by the purchaser for internal research purposes only and distribution is strictly prohibited with
4. SSBI System Biosciences Minicircle DNA and mc iPS Cells Cat SC301A 1 SRMXXXPA 1 User Manual A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 2 111910 contained in this user manual Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 Contents I Introduction and Background ccesceeeeeeeeeeeeeeeaeeeeeeeeeeees 2 A The Minicircle Technology scceecceseteeeeceeeeeeeeeeeeeeees 2 B Minicircle derived iPS cell line 4 Il Protocols eienenn ud a aN AAA NA hee 6 A Transfection of Minicircle DNA for reprogramming 6 B Growing mc iPS CellS ceeceeeeeeeeeeeceeeeeeeeeeeeeesaeeeeeeeeeeeeee 7 III References ss cation an lv ai a iniiai 9 IV Technical Support ceeesceceeeeeseeeeeeaeceeeeeeceaeeesaeeseneessaees 10 V Licensing and Warranty e ssseeseessssiresssrsseserrssrirnnsrrrnssrernsse 10 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual Introduction and Background A The Minicircle Technology Minicircles MC are circular non viral DNA elements that are generated by an intramolecular cis recombination from a parental plasmid PP mediated by C31 integrase The full size MC DNA construct is grown in a special host E coli bacterial strain This strain harbors an Arabinose inducible sys
5. imately 5ug per transfection per well in 6 well plate three times 1 Use Nucleofector Kit R Amaxa and program U 023 according to the manufacturer s instructions 2 Plate transfected cells in 10 cm dishes and culture in DMEM F12 medium Invitrogen supplemented with 10 FBS 3 GFP positive cells can be sorted by flow cytometry 3 days after transfection The sorted cells should then seeded on gelatin coated 6 well plates at 0 5 x 10 cells per well 4 Switch cells to human ESC culture medium 1 day after seeding Refresh culture medium every 2 3 days 5 On days 4 and 6 transfect the cells again with minicircle DNA using Lipofectamine 2000 Invitrogen according to the manufacturer s instructions 6 Colonies with morphologies similar to hESC colonies are clearly visible by day 18 after the initial transfection 7 At day 26 28 after transfection GFP negative mc iPSC colonies can be individually picked for further expansion and analysis Page 6 ver 2 111910 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 The GFP signal should decrease over time correlating with the disappearance of the minicricle DNA A simultaneous increase of the endogenous pluripotency marker expression should also be observed E GFP MC DNA E Oct4 endogenous 1 0 0 5 Fold Change 3 6 9 12 T5 18 21 24 Time days B Growing mc iPS cells Materials Human ESC medium DMEM F12 containing 20 kn
6. ockout serum replacement 2 mM glutamine 1 x 10 M nonessential amino acids 1 x 10 M 2 mercaptoethanol 10 ng ml bFGF and 50 U and 50 ug ml penicillin and streptomycin MEF medium DMEM containing 10 FBS 2 mM glutamine 1x 10 M nonessential amino acids and 50 U and 50 ug ml penicillin and streptomycin ROCK Inhibitor Y 27632 Sigma NOTE This protocol is for growing mc iPS cells on MEF feeder cells These should already be growing before you plate your mc iPS cells 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Processing and culturing mc iPS Cells Upon receiving the vial of human iPS cells quickly thaw the vial of frozen cells in a 37 C water bath Remove the vial from the water bath as soon as the cells are half thawed and sterilize by spraying the outside of the tube with 70 ethanol Add the cells to 10 ml of pre warmed human ES medium in a 15 cm conical tube Pellet the cells by centrifugation at 200 g for 5 min While centrifuging remove MEF medium from the 6 well plate with MEF feeder cells wash the wells twice with 1 ml of DMEM F 12 and add 1ml of human ES medium Discard the supernatant of the tube containing human iPS cells Resuspend the cells with 1 ml of fresh human ES medium supplemented with 10 uM ROCK inhibitor Y 27632 final concentration to decrease spontaneous differentiation and to assist in recovery after freeze thawing Plate the cells
7. out written permission by System Biosciences Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2010 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 11
8. tem to express the C31 integrase and the I Scel endonuclease simultaneously The C31 integrase produces the MC DNA molecules as well as PP DNA from the full size MC DNA construct The PP DNA contains several engineered I Scel restriction sites that ultimately lead to the destruction of the PP DNA but not the MC DNA The difference between MC and standard plasmid vectors is that the MC no longer contains the bacterial origin of replication or the antibiotic resistance markers Sequences within the bacterial plasmid backbone contain signals for methylation and transgene silencing Thus delivering only the minicircles to cells lengthens the expression of the transgene over traditional transient transfections of plasmids SBlI s_ pre made MC LGNSO DNA features easy to transfect molecules that have an extended expression lifespan in mammalian cells to efficiently reprogram somatic cells to the pluripotent state For dividing cells expression of the minicircles lasts up to 14 days For non dividing cells expression of the minicircles drops slightly after the first week but then can continue expressing the transgenes for months Page 2 ver 2 111910 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 pMC LGNSO M _ Arabinose lt PP lt MC Agarose gel showing the induction and production of Minicircle DNA with Lin28 GFP Nanog Sox2 Oct4 Lin28 ha Transfect Source Cells to Induce Pluripotency
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