PhiC31 Reprogrammer & miPSCs, User Manual - Bio
Contents
1. 10 D Validation of PhiC31 Mouse iPS cells cceeeeeee eens 13 IV References sci cech areca ivcientcodes eerecmecddes ataememneicewen 14 We Technical SUD DOM zesse D 15 VI Licensing and Warranty Statement cccceeeeeeeeeees 15 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual Introduction A Background of C31 Integrase Technology The C31 integrase is a site specific recombinase encoded within the genome of the bacteriophage C31 The oC31 integrase mediates recombination between two 34 base pair sequences termed attachment sites att one found in the phage and the other in the bacterial host This serine integrase has been show to function efficiently in many different cell types including mammalian cells In the presence of 6C31 integrase an attB containing donor plasmid can be unidirectionally integrated into a target genome through recombination at sites with sequence similarity to the native attP site These sites are termed pseudo aittP sites 6C31 integrase can integrate a donor plasmid of any size as a single copy and requires no cofactors The integrated transgenes are stably expressed and heritable Transgenes attB donor plasmid attB with transgenes Attachment site Mr do attP PEA farsa genome gt phiC31 Integrase attl attR Transgenes Page 2 ver 3 111312 www systembio com PhiC31 Reprogrammer2. 1 Remove the vial from liquid nitrogen and thaw quickly in a 37 C water bath 2 Remove the vial from the water bath as soon as the cells are half thawed and sterilize the tube by spraying with 70 ethanol 3 Transfer the cells with 10 ml of MEF medium to a 15 mL conical tube and pellet the cells by centrifugation at 200 g for 5 min 4 Discard the supernatant and resuspend the cells with 10 ml fresh MEF medium and plate the cells on gelatin coated plates at seed density of 10 cells cm 5 Incubate at 37 C with 5 COs until the cells reach 80 90 confluency 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual 6 Change medium twice a week or when pH decreases 4 Passaging MEF cells Cells should be split when they reach confluency We recommend splitting the cells based on 0 5x10 4 cells cm 1 2 3 4 5 6 Discard the medium and wash the cells once with PBS Aspirate PBS and add 2 ml per T75 flask of 0 25 trypsin EDTA and incubate for 2 min Add 5 ml of MEF medium and break up the cell clumps by gently pipetting up and down several times Transfer cells into a conical tube and centrifuge at 200 g for 5 min Discard the supernatant and resuspend the cell pellet in 10 ml MEF medium Count the number of cells plate cells at 0 5x10 cells cm and incubate at 37 C with 5 COs 5 Freezing MEF cells 1 2 3 4 5 6 7 Foll
3. x connect supporting life sciences PhiC31 Reprogrammer amp miPSCs User Manual Interest in any of the products request or order them at Bio Connect al Bio Connect B V T NL 31 0 26 326 4450 T BE 32 0 2 503 03 48 m m Begonialaan 3a F NL 31 0 26 326 4451 F BE 32 0 2 503 03 27 6851 TE Huissen E info bio connect nl o The Netherlands W www bio connect nl SSBI System Biosciences PhiC31 Reprogrammer amp miPSCs Catalog s FC300A 1 SC211A 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 1 11 14 12 contained in this user manual PhiC31 Reprogrammer amp miPSCs Cat s FC300A 1 SC211A 1 Contents I AL ROCUICHON a ictetat a uttan a A 2 A Background of 6C31 Integrase Technology 006 2 B C31 Reprogramming Vector Maps amp Details 3 G Listof COMPONENIS ccee eee tee eee 4 ll Protocol for Deriving Mouse Induced Pluripotent Stem Cells using the 6C31 Reprogrammer on MEF SOUC CONS aiaa a a a 5 A Mouse Induced Pluripotent Stem Cell Generation 5 III Non Viral C31 Integrase derived Mouse iPS Cells 6 A C31 Generated Mouse Induced Pluripotent Cell Line 6 B Culture Conditions for MEF Feeder Cells cceccceeeeees 6 C Growth Conditions for PhiC31 Mouse iPS cells
4. User Manual phiC31 Integrase Expression Vector Cale Origin phiC31 Integrase Expression Plasmid Cat FC200PA 1 Kan 4 9 kb phiC31_Integrase Kan Promoter bGHpA C List of Components List of Components for FC300A 1 Reprogramming Kit Each Non Viral PhiC31 Integrase Mouse iPSC Reprogramming Kit includes 10ug of plasmid DNA for FC200PA 1 and FC305A 1 PhiC31 Integrase Plasmid FC300A 1 FC200PA 1 amp PhiC31 10ug each Reprogrammer FC305A 1 List of Components for C211A 1 PhiC31 Mouse iPS Cells Each iPS cell line set comes as one vial of MEF derived PhiC31 generated Mouse iPS cells and one vial of MEF at 2 x 10 cells each Page 4 ver 3 111312 www systembio com PhiC31 Reprogrammer amp miPSCs Cat s FC300A 1 SC211A 1 Mouse iPS Cell Line Set K SC211A 1 IMEF amp PhiC31 miPSCs 2x105 cells each The product is shipped on dry ice and should be immediately stored in the gas phase of liquid nitrogen In general iPS cells are challenging to culture and should only be operated by researchers experienced in the intricacies of mouse embryonic stem mES cell culture The methods for culture are nearly identical to mES cell culture although more careful maintenance will be required The MEF cells provided in the iPSC kit are meant to be used as source cells only and not as feeder cells for culturing iPS cells Appropriate feeder cells for mouse iPS cell culture can be obtained from commercial sources
5. amp miPSCs Cat s FC300A 1 SC211A 1 Fig 1 Schematic of the C31 Integrase mediated recombination of donor plasmid sequence into pseudo attP sites in host genome B C31 Reprogramming Vector Maps amp Details The classic Oct3 4 Sox2 KIf4 and cMyc factors have been adapted to the integrase system The pCOBLW phiC31 reprogramming vector catalog FC305A 1 features the iPSC factors driven by a potent CAGs promoter with the factors separated by P2A elements HS4 insulators flank the expression cassette and a functional attB site directs the integration of the entire plasmid into pseudo attP sites in any genome The vector is engineered with FRT sites for later excision using standard FLP recombinase methods After co transfection of the reprogramming vector with the phiC31 expression plasmid catalog FC200PA 1 and integration you can monitor for successful transfection with GFP fluorescence and select for positive colonies using the built in Neo marker The reprogramming iPSC factor donor vector and the phiC31 expression plasmid maps are shown below IPSC Reprogramming Donor Vector CAG prorneber eel _ KarnineerR s q Te P iih swat vb m CTA4 promoter 7 h S mPGKI re b b promoier l i a T E l PaA Y yA OBLW 4 SV40pA pc mSouz i phiC31Reprogrammer Cati FC305A 1 Ah cHsa 14 5 kb 1 insulator i aHa nsulator 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI
6. colonies will be visible starting from 8 to 12 days and should be picked between 20 and 26 days for further expansion See Section Ill for additional protocol details on MEF feeder cell generation and iPSC culture techniques Non Viral 6C31 Integrase derived Mouse iPS Cells A C31 Generated Mouse Induced Pluripotent Cell Line Mouse induced pluripotent stem cells IPSCs Cat SC211A iPSC were generated by nucleofecting genetically unmodified C5 7BL 6 mouse embryonic fibroblasts with the pCOBLW C31 Integrase Mouse Reprogramming Donor plasmid Cat FC305A 1 and the C31 Integrase expression plasmid Cat FC200PA 1 for stable integration into the mouse genome Karow 2011 The Reprogramming Donor plasmid encodes the four murine transcription factors Oct4 Sox2 Klf4 and c Myc that have been shown to induce the reprogramming of somatic cells to a pluripotent state See Section II of this User Manual for more details on the reprogramming protocol used to make this cell line Due to the specificity of recombination using the C31lntegrase system the iPSCs are expected to have on average one copy of the OSKM plasmid per cell which reduces the risk of insertional mutagenesis effects integration analysis data available on Pg 18 The cells were derived using morphological selection criteria and GFP expression without drug selection When cultured under standard mouse ES cell culture conditions the morphology of SBI mouse iPSCs are identical to that
7. defined factors Cell 126 663 676 Takahashi K et al 2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 131 861 72 Park IH et al 2008 Reprogramming of human somatic cells to pluripotency with defined factors Nature 451 141 6 Baker Monya 2007 Adult cells reprogrammed to pluripotency without tumors Nature Reports Stem Cells 2007 12 11 Page 14 ver 3 111312 www systembio com PhiC31 Reprogrammer amp miPSCs Cat s FC300A 1 SC211A 1 Nakagawa M et al 2008 Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts Nature Biotechnology 26 101 106 Okita K et al 2007 Generation of germline competent induced pluripotent stem cells Nature 448 313 7 Yu J et al 2007 Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells Science 318 1917 1920 V Technical Support For more information about SBI products or to download manuals in PDF format please visit our website http www systembio com For additional information or technical assistance please call or email us at tech systembio com 650 968 2200 VI Licensing and Warranty Statement Limited Use License Use of the human and mouse iPS cells 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
8. the AP Detection Kit Millipore Integration Analysis A major advantage of using the phiC31 integrase system is that copy number control is in your hands The majority of clones that undergo integration events have single insertions To verify the copy numbers in your targeted cells you can perform Southern blot analysis on their genomic DNA Shown below is an example of mouse genomic integration analyses using separate restriction digests with Eco01091 and EcoRV with 10ug gDNA The reason for two separate enzyme digests in to assess the copy number status The probe used in these Southern blot analyses was for GFP present in the 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual pCOBLW iPSC integrase vector The data below show the copy number status for 16 different iPSC lines and demonstrate that 14 out of 16 lines show single insertions 88 MEF iPSC Clones Generated with pCOBLW Eco01091 MEF iPSC Clones Generated with pCOBLW m eo een kb 10 a ki 8 G a 0 wi i EcoRV IV References Karow M et al 2011 Site specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA Stem Cells 29 11 1696 704 Rossant J 2007 Stem cells The magic brew Nature 448 260 262 Takahashi K and Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by
9. 0A 1 SC211A 1 2 Maintenance of mouse iPS cells It is important not to keep mouse iPS cells in culture for long period of time without passaging to maintain the pluripotency 1 2 3 4 5 6 7 8 9 Aspirate the medium and wash the cells twice with 1 ml PBS Remove PBS completely and add 0 7 ml of 0 05 trypsin EDTA solution and incubate at 37 C for 10 min While incubating remove a 6 well plate of feeder cells mitomycin C treated MEFs Aspirate the medium and add 2 ml of mouse iPSC medium to each well Remove the plate containing mouse iPS cells from the incubator and swirl to dislodge the cells from the bottom of the plate Add 2 ml of mouse iPSC medium and suspend the cells by pipetting up and down to single cell suspension Transfer the cell suspension to a 15 ml conical tube and spin the cells at 200 g for 5 min Add 2 ml of iPSC medium to the plate and suspend the cells by pipetting up and down to single cell suspension Distribute 0 2 ml of the mouse iPS cell suspension to each well of the 6 well plate Right after plating iPS cells gently swirl the plate back and forth and side to side and incubate at 37 C with 5 CO2 until the cells reach 80 confluency The mouse iPSC media must be changed every day and mouse iPS cells subcultured 1 10 every 2 3 days Track the passage number of the iPS cells 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User M
10. anual 3 Freezing mouse iPS cells 1 2 3 4 5 6 7 8 9 Grow cells to the exponential phase in a 6 well plate Aspirate the medium and wash the cells twice with 2 ml of PBS Aspirate the medium and wash the cells twice with 2 ml of PBS Add 0 7 ml 0 05 trypsin EDTA and incubated for 10 min at 37 C Add 2 ml of mouse iPSC medium and suspend the cells by pipetting up and down to single cell suspension Transfer the cell suspension to a 15 ml conical tube count the number of cells and spin the cells at 200 g for 5 min Discard the supernatant and resuspend the cells with mouse iPSC medium to the concentration of 1x10 cells per ml Add equal volume of 2x freezing medium and aliquot it at 0 5 ml per vial Put the vials in a cell freezing container and store them at 80 C overnight 10 Transfer the vials to a liquid nitrogen tank for long term storage Page 12 ver 3 111312 www systembio com PhiC31 Reprogrammer amp miPSCs Cat s FC300A 1 SC211A 1 D Validation of PhiC31 Mouse iPS cells White Alkaline Phosphatase Oct4 Ca r gt e e Sox2 Nanog SSEA 1 Stem cell markers for Oct4 Sox2 SSEA 1 and Nanog were determined by immunocytochemistry using primary antibodies for SSEA1 Millipore Oct4 Abcam Sox2 Abcam and Nanog Abcam followed by Alexa Fluor fluorescent labeled secondary antibodies Invitrogen Detection of Alkaline Phosphatase activity was performed using
11. 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 e 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 e The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI e This Product should be used in accordance with the NIH guidelines developed for stem cell research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in th
12. is manual 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 SBs 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 2012 System Biosciences SBI Page 16 ver 3 111312 www systembio com
13. of mouse ES cells The cells also express the pluripotency markers SSEA 1 Nanog Sox2 and Oct4 and demonstrate strong endogenous AP activity Mouse iPS cells from SBI are provided at passage 10 and can be passaged 50 times before differentiation B Culture Conditions for MEF Feeder Cells Mouse iPS cells should be grown on a feeder cell layer Appropriate feeder cells for mouse iPS cell culture can be obtained from commercial sources or generated through Mitomycin C treatment of mouse embryonic fibroblasts MEFs Page 6 ver 3 111312 www systembio com PhiC31 Reprogrammer amp miPSCs Cat s FC300A 1 SC211A 1 1 Required media and reagents Reagent Information MEF Medium DMEM containing 10 FBS 2 mM glutamine 1x10 M nonessential amino acids and 50 U and 50 ug ml penicillin and streptomycin 2x Cold 20 DMSO and 80 FBS Freezing Media Mitomycin C 1 mg ml solution 2 Gelatin treatment of plates for MEF feeder cells 1 Add enough sterile autoclaved 0 1 gelatin to cover the bottom of the wells Approximate amounts 10cm plate 5 ml 6 well 1 5 ml well 24 well 0 5 ml well 96 well 100 ul well 2 Incubate the gelatin coated dishes for at least 15 min at 37 C 3 Aspirate excess gelatin solution before use 3 Thawing MEF cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells Cells should be plated at a minimum cell density of 104 cells cm
14. or generated through Mitomycin C treatment of mouse embryonic fibroblasts MEFs See protocol on Pgs 9 12 for more details on generating feeder cells ll Protocol for Deriving Mouse Induced Pluripotent Stem Cells using the C31 Reprogrammer on MEF source cells A Mouse Induced Pluripotent Stem Cell Generation The following protocol has been optimized for mouse embryonic fibroblasts MEF cells as described in Karow M et al Other source cells may require transfection optimization for efficient reprogramming In general reprogramming requires a single transfection of approximately 3 ug per well in a six well plate 1 Use the MEF nucleofector kit Lonza and the program T 20 according to the manufacturer s instructions 2 Nucleofect with 3 ug total DNA using the PhiC31 Integrase Plasmid FC200PA 1 amp PhiC31 Reprogrammer Donor Plasmid FC305A 1 at a 1 1 ratio by mass The Neon transfection system from Life Technologies has also been demonstrated to work with similar efficiency 3 After nucleofection plate the cells in a well of a six well plate using MEF media Change media each day with MEF media 4 On day two transfer the cells to a 0 1 gelatin coated10 cm dish containing irradiated or mitomycin C treated MEF feeder cells Switch to Mouse iPSC growth media cat no SC200M 1 Change the media every other day 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 5 GFP positive
15. ow steps 1 4 from the Passaging MEF cells protocol above Discard the supernatant and resuspend the pellet in MEF medium Count the number of cells and adjust the cell suspension to 4 x 10 cells ml Add equal volume of cold 2X Freezing Media to the cell suspension Aliquot 1 ml of suspension into each cryovial 2 x 10 cells vial Place the vials in a cell freezing container and keep itat 80 C overnight Transfer the vials to a liquid nitrogen tank for long term storage Page 8 ver 3 111312 www systembio com PhiC31 Reprogrammer amp miPSCs Cat s FC300A 1 SC211A 1 6 Mitomycin C treatment of MEF Mitomycin C acts to halt the division of MEF cells so that they can be used as a feeder layer for miPS cells MEF cells should be at confluence when treated with mitomycin C 1 2 3 4 5 6 7 Add 6 ml of fresh MEF medium contain 50 ul of mitomycin C solution 1 mg ml to one T75 flask of confluent MEF cells and swirl it briefly The final concentration of mitomycin C is 8 ug ml Incubate at 37 C for at least 3 hrs Aspirate the mitomycin C containing medium off the cells and wash the cells twice with 10 ml PBS Aspirate PBS and add 1 ml of 0 25 trypsin EDTA swirl to cover the entire surface and incubate for 2 min at room temperature Add 5 ml MEF medium and break up the cells to a single cell suspension by pipetting up and down Count the number of cells Seed the cells on gelatin coated dishe
16. s 3 x 10 cells per 100 mm dish or 5 x 10 cells per well of a 6 well plate Cells should be ready to use by the next day 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual C Growth Conditions for PhiC31 Mouse iPS cells 1 Required Media and Reagents Reagent Information Mouse iPSC SC200M 1 Growth Medium 2x Cold Freezing 20 DMSO and 80 ES FBS Media Trypsin EDTA GIBCO 1 Thawing Mouse iPS cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells 4 5 6 Remove the vial from liquid nitrogen and thaw quickly 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 with 70 ethanol Transfer the cells with 10 ml of mouse iPSC medium to a 15 mL conical tube and pellet the cells by centrifugation at 200 g for 5 min While centrifuging remove MEF medium from the feeder cell plates and wash the wells twice with DMEM Then add 1 ml of mouse iPSC Medium Discard the supernatant from the mouse iPS cells and resuspend cells with 1 ml fresh mouse IPSC medium Plate the cells on MEF feeder cells in a 6 well plate Incubate at 37 C with 5 CO until the cells reach 80 confluency The mouse iPSC media must be changed every day Mouse iPS Cells Express GFP Page 10 ver 3 111312 www systembio com PhiC31 Reprogrammer amp miPSCs Cat s FC30
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