Minicircle iPSC Technology User Manual
Contents
1. 3 Aliquot into appropriate amount according to usage and store the aliquots at 4 C Coating Plates with Matrigel Matrigel Cat 354277 BD should be aliquoted and stored at 80 C for long term use 1 Thaw matrigel on ice until liquid Dilute matrigel 1 50 to 1 100 with pre chilled KO DMEM F 12 2 Immediately use the diluted matrigel solution to coat tissue culture treated plates For a 6 well plate use 0 8 mL of diluted matrigel solution per well and swirl the plate to spread the matrigel solution evenly across the surface 3 Let the coated plate stand for 1 h at 37 C or overnight at 4 C If plate has been stored at 4 C allow the plate to incubate at 37 C for 30 minutes before removing the matrigel solution Thawing Cryopreserved hiPSCs 1 Quickly thaw the hiPSCs in a 37 C waterbath by gently shaking the cryovial continuously until half thawed Remove the cryovial from the waterbath and spray with 70 ethanol to sterilize Page 8 ver 2 022013 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 2 Transfer the contents of the cryovial to a 15 mL conical tube Add 5 mL warm mTeSR1 dropwise to the tube gently mixing as the medium is added 3 Centrifuge cells at 200 x g for 5 minutes at room temperature 4 While centrifuging remove the matrigel solution from a coated tissue culture 6 well plate Add 1 mL of warm mTeSR1 containing 10 uM ROCK inhibitor to one well of 6 well plate 5 After2. Add the rinse to the 15 mL tube 8 Centrifuge the 15 mL tube containing the aggregates at 200 x g for 5 minutes at room temperature 9 Aspirate the supernatant Resuspend pellet in mTeSR1 containing 10 uM ROCK inhibitor by gently pipetting and ensure that cells are maintained as aggregates 10 Plate the hiPSC aggregates with mTeSR1 onto a new plate coated with matrigel Remove matrigel solution before plating f the colonies are at an optimal density the cells can be split every 5 7 days using 1 6 to 1 10 splits 12 Place the plate into the 37 C incubator and move the plate in quick side to side forward to back motions to evenly distribute the clumps within the wells Culture the cells at 37 C with 5 CO2 and 95 humidity 13 Change medium daily Cryopreserving hiPSCs 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual 1 Prepare freezing medium which contains 90 FBS 10 DMSO and 10 uM ROCK inhibitor 2 Perform step 1 8 from Passaging hiPSCs Grown under Feeder free condition 3 Gently aspirate the supernatant taking care to keep the cell pellet intact 4 Gently resuspend the pellet in freezing medium taking care to leave the clumps larger than would normally be done for passaging 5 Transfer 1 mL of clumps in freezing medium into each labeled cryovial 6 Place vials into an isopropanol freezing container and place the container at 80 C to 150 C overnight 7 Tran
3. 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 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 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 2013 System Biosciences SBI All Rights Reserved Page 12 ver 2 022013 www systembio com
4. 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 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 without 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
5. Arabinose lt MC User Manual 9493 Mei 2 Plasmid Backbone 7466 _ 3805 32 1Sce1 sit 1 CMV promoter 1065 1345 Lin28 1536 2162 Kanamycin Resistance PMC_LGNSOmap 7465 6905 9605 bp GFP 2235 2687 ap 6487 6525 E335 katet 2 hNanog 2367 3881 MC LGNSO 774 6478 hOct4 4377 6053 NSox2 3954 4904 ver 2 022013 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 B 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 approximately 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 hESC 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 C
6. DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 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 Phase Contrast MK wb Alkaline Phos RT PCR Oct4 endo Sox2 endo Nanog endo Lin28 endo ERAS DPPAS ECAT1 TDGF1 wech CO L 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual The mc iPSCs also demonstrate multiple lineage potential Undifferentiated U Differentiated D Page 4 ver 2 022013 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 Protocols A Minicircle Production The following protocol requires that you use both the pMC LNSO parental construct in combination with the ZYCY10P3S2T E coli strain This strain has been specifically engineered to express the C31 integrase and l Scel endonuclease upon arabinose induction Materials Component Cat Source LB Agar Kanamycin L1025 Teknova 50 plates 20 pk 20 Arabinose solution A2010 Teknova 50ml Kanamycin Solution 50mg mL 25 mL K2125 Tekno
7. 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 Backoround 2 A The Minicircle Technology 2 D Minicircle derived iPS cell line 2 0 0 eee ee eeeeeeeeeteeeeeeenaes 3 INe gt POLO COIS EEE cttecuh ia teeie bal inccbkbeuih ei ulead babieetand ecebiiatlons 5 Minicircle Production 0 cccccecceeeeeeeeeeeeeeeeeeeeeeeteeneeeeeeaeees 5 B Transfection of Minicircle DNA for reprogramming 7 Growing mc iPS cells in Feeder Free Medea 8 III Ee 10 IV Technical Gupport 11 V Licensing and Warranty cccceeeseeeeeeeeeeeeeeeeeeeeeeeneeeeeeaaes 11 888 266 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 system to express the C31 integrase and the I Scel endonuclease sim
8. centrifugation aspirate the medium from 15 mL tube Gently resuspend the cell pellet in 1 mL mTeSR1 with 10 uM ROCK inhibitor taking care to maintain the cells as small aggregates 6 Transfer the medium containing the cell aggregates to the matrigel coated 6 well plate 7 Place the plate into the 37 C incubator and move the plate in quick side to side forward to back motions to evenly distribute the clumps within the wells Culture the cells at 37 C with 5 CO2 and 95 humidity 8 Change medium daily Check for undifferentiated colonies that are ready to passage approximately 7 10 days after thawing Passaging hiPSCs Grown under Feeder free condition 1 Look under microscope to identify regions of differentiation Mark the differentiated colonies using lens marker on the bottom of the plate 2 Remove regions of differentiation by scraping with a pipette tip or by aspiration 3 Aspirate medium from the hiPSC culture and rinse with DPBS 2 mL well 4 Add 0 5 mL per well of accutase Cat SCR005 Millipore diluted 1 1 with DPBS before use Let it stand at room temperature for 1 2 minutes 5 Remove accutase and gently rinse each well 2 3 times with 2 mL of DMEM F 12 per well to dilute away remaining enzymes 6 Add 2 mL well mTeSR1 and scrape colonies off with a cell scraper 7 Transfer the detached cell aggregates to a 15 mL conical tube and rinse the well with an additional 2 mL mTeSR1 to collect any remaining aggregates
9. olonies 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 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual 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 9 Wis T 23 24 Time days C Growing mc iPS cells in Feeder Free Media Materials e mTeSR 1 basal media and supplements Cat no 05850 StemCell Technologies e Human ES medium DMEM F12 containing 20 knockout serum replacement 2 mM glutamine 1 x 10 4 M nonessential amino acids 1 x 10 4 M 2 mercaptoethanol 10 ng ml bFGF and 50 U and 50 mg ml penicillin and streptomycin e Matrigel Cat no 354277 BD Biosciences e Accutase Cat no SCR005 Millipore e ROCK Inhibitor Y 27632 Cat no Y0503 Sigma Preparation of Feeder free medium 1 Thaw mTeSR1 5X Supplement Cat 05850 STEMCELL Technologies at room temperature or overnight at 4 C 2 Add the 100 mL of thawed 5X Supplement to 400 mL Basal Medium for a total volume of 500 mL aseptically Mix well Filter through a 0 2 um low protein binding filter if desired
10. sfer to a liquid nitrogen tank the next day 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 Page 10 ver 2 022013 www systembio com Minicircle DNA and mc iPS Cells Cats SC301A 1 SRMXXXPA 1 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 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 Le
11. ultaneously 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 SBI 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 pMC LGNSO M _ Arabinose lt Pp lt Mc Agarose gel showing the induction and production of Minicircle DNA with Lin28 GFP Nanog Sox2 0ct4 Lin28 o a e G O Transfect Source Cells to Induce Pluripotency MC LGNSO Minicircle Reprogramer D Catalog SRM100PA 1 Create Nonviral iPS Cells Qo 4 Page 2 ver 2 022013 www systembio com Minicircle
12. va Terrific Broth Complete T7055 Teknova L8000 Teknova PureLink HiPure Plasmid Filter Purification Kit K2100 14 Invitrogen Procedure 1 On day 1 in the morning grow cells from a plasmid transformed colony in 5 ml LB containing 50 ug ml kanamycin at 37 C with shaking at 250 rpm On day 1 in the evening grow an overnight culture by combining 100 ul of the culture in step 1 per 400 ml of fresh TB Terrific Broth containing 50 ug ml kanamycin in a 2 liter flask and culture at 37 C with shaking at 250 rpm For scaling up the protocol keep the ratio of flask size to O N culture volume at 5 1 vol vol On day 2 in the morning the OD600 of the overnight culture will be between 4 and 5 Prepare a Minicircle Induction Mix comprising 400ml LB 16 ml 1N NaOH and 400 ul 20 L arabinose Combine the Minicircle Induction Mix with the overnight culture and incubate at 30 C with shaking at 250 rpm for 5 hours or longer Pellet the bacteria and use a PureLink HiPure Plasmid Filter Purification kit Invitrogen Cat K2100 14 to isolate minicircle DNA according to manufacturer s protocol The gel image below shows uninduced minus arabinose and the LGNO minicircle preps digested with Mfel The parental construct generates 2 bands of 6 7kb and 3 2kb The Minicircle MC DNA produces a single band about 6kb 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI pMC LGNSO Kb Page 6
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