PiggyBac Transposon Vector System User Manual
Contents
1. PiggyBac Dual Promoter Vector Formats ype Terminal Repeat Promoter Catalog Marker PB5108 1 PB5118 1 c AV PBS128 1 AFP TMV PBS13B 1 _ GFF PUrO CMV Pe5148 1 RFP ePuro Mocy 1P87138 1 GFP Pura put PiggyBac Single Promoter Vector Formats E Terminal fiepeat Page 13 System Biosciences SBI User Manual shRNA expression vectors 5 PB Terminal Amp Repeat Core Insulator puc ori shRNA Vectors EF 1c or CMV Cat PBSISOSA 1 CMV ypo Cat PBSI506A 1 EF1 Terminal Repeat a P pio p SIRNA template Insert gt marn shRNA m Dicer RNA arte re PiggyBac Transposase expression vectors ina a Excision Only Super PiggyBac PiggyBac Transposase CMV Transposase CMV Promoter Expre ssion Vector Promoter i l cat PB220PA 1 Sad D9 SV40 late 16s Intron SV40 late Super PiggyBac Excision only Transposase PiggyBacTransposase Page 14 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx F PiggyBac copy number kit To determine the number of piggyBac transposon integrations SBI has developed a qPCR based system to measure the copy number of transposons in genomes relative to a genomic counting primer set cat PBC100A 1 The UCR1 primer mix counts the number of genomes and the PBcopy primer set measures the number of piggyBac transposons present in the transgenic genome The PBcopy primer set is compatible with all of SBI s2. o 1X cell Lysis buffer Materials required but not included o 2X SYBR green mastermix o qPCR instrument o 1x PBS PROTOCOL Genomic DNA preparation from cells in a 12 well culture plate 1 Remove media and wash cells with 1mL 1x PBS 2 Remove all of PBS and add 250uL Lysis buffer to each well You may freeze cells at 80 C until ready It is recommended to perform one freeze thaw cycle with the Lysis buffer to ensure complete cellular lysis then thaw the plate at room temperature Page 16 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx a Pipet cells up and down to detach them and remove cell clumps Transfer cell lysates in a 1 5ml eppendorf tube Heat lysates at 95 C 2 minutes Centrifuge at 13 000 rpm 2 minutes in a standard microcentrifuge to pellet debris Transfer supernatant to a new tube and place tubes on ice or store at 20 C until ready to proceed our N qPCR set up for copy number quantitation 384 well plate per reaction for PB detection water 4 75 uL 2x SYBR Green 6 25 uL PB copy primer mix 5uM 0 5 uL lysate lt 500ng DNA 0 5 uL per reaction for genomic DNA detection water 4 75 uL 2x SYBR Green 6 25 uL 25x UCR1 primer mix 0 5 uL lysate lt 500ng DNA 0 5 uL 1 Prepare 2 mastermixes of water 2x SYBR green and primers one for UCR1 and one for PBcopy primers for all samples including duplicates e g double all reaction ingredients and multiply by
3. 6 cells well NOTE If the cells have been previously growing under selection conditions e g in Puro Neo or other antibiotic markers please passage the cells 1x or 2x in absence of selection marker prior to using the Excision only transposase 2 For one well of a 6 well dish 1 x 106 cells Combine 0 2 ug PiggyBac Excision Only Transposase vector PB220PA 1 8 0ul SBI s PureFection transfection reagent 50 ul of serum free DMEM Vortex 15 30 seconds Let stand 15 minutes at room temperature to allow PureFection DNA complexes to form 5 Add drop wise to cells culture and swirl to disperse 6 Assay for transposon removal in target cells 72hrs after transfection of the plasmid Transposon copy number after excision in target cells can be assayed by SBI s Piggybac Copy Number Kit Cat PBC100A 1 Details on Section F pg 15 Page 8 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx Sample Re excision data Shown below is an example of the excision only piggyBac transposase lack of integration activity and its excision capabilities The left panel shows the comparison between the integration activities of Super PiggyBac transposase PB210PA 1 and the inability of the Excision only piggyBac transposase PB220PA 1 to integrate transposons The panel to the right shows the seamless excision activity of PB220A 1 An engineered GFP expression cassette with a piggyBac transposon integrated right i
4. 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 SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI 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 this 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 othe
5. piggyBac transposon vectors The copy number of piggyBac transposons is calculated using the cycle threshold Ct values of the UCR1 signals relative to the PBcopy signals The PBC100A 1 copy number kit comes with primers and cell lysis buffer reagents enough for 20 copy number determinations NOTE Your cells must be passaged at least once before performing this copy number measurement to ensure that residual non integrated piggyBac transposon plasmid will not interfere with the qPCR Ratios of PB transposon to PB transposase can be done to adjust the copy number of integrations First average the Ct values for the UCR1 and PBcopy measurements The Delta delta AA Ct calculation 2 avg PBcopy Ct avg UCR1 Ct The copy number is determined by dividing the AA Ct by 2 as there are 2 copies of the UCR sequence per genome 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual Transposon Traesposase Pheopy UCR avg ra copyt lng ng ave a AACt 2 t t 100 100 23 82 2843 24 47 12 TE 100 1988 2474 29 00 15 500 100 19 05 2469 49 76 25 700 100 18 77 2497 73 29 37 d l as 100 eis 249 0 92 40 y 0 0352 7 4258 R 0 9388 W w O w vo uw Number of Copies per Genome 0 200 400 600 800 1000 PB Transposon added ng PBase held constant at 100ng Materials provided in kit o 25x UCR1 primer mix o 25x PBcopy primer mix
6. PureFection transfection reagent 50 ul of serum free DMEM 2 After transfection of cells split the cells into multiple wells of a 6 well plate or a single 10cm plate and grow them to confluency 3 Apply puromycin selection to establish positively transposed cells 2 5ug ml for 3 5 days 3 Change medium maintain puromycin selection and titrate in the Cumate induction solution 10 000x high concentration cat PBQM100A 1 We recommend starting with a 1x concentration equivalent to 30ug ml cumate and increasing the cumate up to 10x equivalent to 300ug ml to test the best induction in your model cell system TO KEEP THE SWITCH ON maintain the appropriate level of Cumate in the media after passages 4 The induction should be immediate and you should be able to visualize induction of the GFP marker within 2 3 days 5 TO TURN BACK OFF simply rinse the cells once with fresh media and add back fresh media WITHOUT any Cumate The cumate switch should turn off immediately and you should see the GFP levels reduce over 2 3 days Page 10 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx Sample Induction data The PiggyBac Cumate Switch is absolutely leak proof 5 PB Terminal Amp Repeat Core Insulator puc ori Cumate ae Cumate Switch Switch Terminal Inducible Vector Pee Ks Repeat lt cat PBQMB12A 1 by Core insulator sva0 poly A CymR EF Te 888 266 5066 Toll Free 650 968 2200
7. SBI System Biosciences PiggyBac Transposon Vector System Cat PBxxx 1 User Manual Store kit at 20 C on receipt 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 contained in this user manual PiggyBac Transposon System Cat PBxxx Contents Protocol A Overview a cc cccescsececeensececsansesecsareeteceaneeteneanentese 3 B PiggyBac transposition protocols oe 4 C PiggyBac re excision protocols cece 8 D Inducible PiggyBac Cumate switch system sss 9 E PiggyBac Vector Maps ooo ceeseesesseereneeeren 13 F PiggyBac Copy Number qPCR Kit 15 G Technical Support sss 18 ll Licensing and Warranty Statement 19 Transposagen s PiggyBac Vectors are Distributed by System Biosciences The PiggyBac Genetic Modification System enables researchers to e Alter the genomes of numerous animal species with a simple transfection e Reprogram somatic cells into induced pluripotent stem iPS cells e Perform highly efficient and cost effective non viral gene delivery e Reverse genomic modifications with footprint free transposon removal The piggyBac DNA transposon technology is already being utilized in multiple research areas such as gene therapy regenerative medicine cell line engineering and animal model creation 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manu
8. al Researchers in academia and the pharmaceutical and biotechnology industries can now purchase piggyBac vectors produced with SBI s high standards of manufacturing and quality control Transposagen s piggyBac technology will be paired with SBI s existing leading edge technologies to further broaden the utility of piggyBac SBI provides piggyBac in custom vectors and cell lines for customers Transposagen and SBI have agreed to collaborate in the production of custom transgenic rats The collaboration will combine SBI s genomic tools and expertise with Transposagen s piggyBac and rat spermatogonial stem cell technology Researchers will now have access to specialized transgenic rats incorporating SBlI s reagents such as species specific RNAi MicroRNAs or anti MicroRNAs Rat models with inducible gene expression can also be produced using SBI s SparQ cumate switch system the first inducible system that can be induced with a small molecule that can readily cross the blood brain and blood testis barriers Page 2 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx The PiggyBac Transposon System A Overview The PiggyBac PB transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a cut and paste mechanism During transposition the PB transposase recognizes transposon specific inverted terminal repeat sequences ITRs located on both ends of the trans
9. er PiggyBac transposase transient expression vector PB210PA 1 and PB513B 1 PB531A 1 were co transfected into Human 293 cells and Puromycin selection applied for 7 days 2ug ml The transposed cells were Puro resistant GFP positive and RFP positive Easily make novel cell lines and animal models with PiggyBac multiplexed transpositions piggyBac Transposase Terminal Amp Repeat Core insulator Dual Promoter PB513B 1 CMV 3 PB Terminal Repeat mcs Core insulator EF la 5v40 poly A GEP Puro puc ort PiggyBac Single Promoter PBS31A 1 3 PB Terminal gt Repeat Core 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual C PiggyBac Transposon Re excision Integrated piggyBac transposons can be successfully removed footprint free using the engineered Excision only piggyBac transposase expression construct PB220PA 1 This engineered piggyBac transposase has two major advantages 1 Integration deficiency and 2 Retains Excision capabilities To seamlessly remove piggyBac transposons you simply transfect your stably transposed cell line with the Excision only piggyBac transposase expression construct to mobilize and excise the integrated transposons Protocol for Piggybac Excision In Target Cells 1 Transfer cells containing the piggybac transposon into a single well of a 6 well plate and grow to confluency 1 x 10
10. ex 15 30 seconds Let stand 15 minutes at room temperature to allow PureFection DNA complexes to form 6 Add drop wise to cells culture and swirl to disperse 7 The PiggyBac transposase activity will terminate after 72 hours but will integrate the transposon vector into genomes 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 8 Check for positive integrations after 3 days We recommend using a 1 2 5 or 1 5 ratio of transposase to transposon vector ratio for transfections Single Transposition data The Super PiggyBac transposase transient expression vector and PB513B 1 were co transfected into HeLa cells and puromycin selection applied for 10 days 10ug ml Cells efficiently transposed were Puro resistant and GFP positive Human 293 cells were transfected with the Super PiggyBac transposase transient expression vector and PB531A 1 The cells were photographed after 7 days virtually all cells were RFP positive PB Transposase PB Transposase S P6 Terminal Repeat PiggyBac Dual Promoter PB513B 1 7P amp Terminal Repeat ineuiistor SV40 PAYA GFP Puro Pvrornn ietection Oyq ied 3 days 5 P8 PB Transposase PB Transposase Terminal Repeat Single Promoter PBS31A 1 Page 6 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx Double Transposition data Integrate multiple PB vectors simultaneously The Sup
11. n the center of the GFP gene knocks out GFP expression When the integrated piggyBac transposon is successfully excised using PB220PA 1 the reading frame is seamlessly re joined and GFP is positively expressed The Excision only piggyBac transposase is also not able to integrate transposons just remove them Excision Capable PBx Integration Deficient PBx GFPa PB GFPb Vector Super piggyBac Excision piggyBac GFP a GFP b o Eze Excision piggyBec 2 sd Ny mao z Integrated PiggyBac Vecto Notransposase Excision piggyBec hromoanenal fuk 2 5 nese wer gt P Fain lt e my a Footprint tree removal D Inducible PiggyBac Cumate Switch All in one inducible vector is leak proof The inducible PiggyBac vector features the ultra tight cumate switch combined with the EF1 CymR repressor T2A Puro cassette to establish stable cell lines Expression of your cDNA or microRNA of interest can be switched on simply by adding cumate to the cells The all in one single vector format offers superior control of induction with no background leakiness 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Co transfect your target cells with the transposase and Cumate switch PiggyBac vectors Example below is for one well of a 6 well plate 1 Combine 0 5 ug PB Cumate switch Transposon vector clone PBQM812A 1 0 2 ug PiggyBac Transposase vector PB210PA 1 8 0ul SBI s
12. outside US Page 11 System Biosciences SBI User Manual PiggyBac Cumate Switch is Fully Titratable No Cumate 25ug miCumate 50 ig miCumate 100ug ml Cumate f ON ON ON PiggyBac Cumate Switch can be Turned OFF Remove Cumate to turn OFF an ON Page 12 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx E PiggyBac Transposon System Vectors cDNA and microRNA expression vectors The PB51x Dual Promoter Series High levels of expression from the CMV promoter most cell types and PB713B 1 features the MSCV promoter active in stem cells The multiple cloning site MCS located downstream of a promoter allows for convenient cloning of your gene or microRNA of interest Downstream of your expression cassette is an EF1alpha promoter driving the expression of either the Puro GFP RFP GFP Puro or RFP Puro markers The entire cassette is flanked by genomic insulator elements for stabilized expression and PiggyBac Inverted Terminal repeats for mobilization and integration The PB53x EF1 Series with IRES Co expressed markers SBI s PiggyBac IRES expression vectors use the EF1alpha promoter to drive expression of your cDNA or microRNA cloned into the MCS along with IRES mediated co expression of the marker The markers available for this PiggyBac vector series include GFP RFP and Neo 888 266 5066 Toll Free 650 968 2200 outside US 5 P8 Terminal flepeat
13. poson vector and efficiently moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites The powerful activity of the piggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes No cargo limit and is Reversible The unique features of piggyBac transposons are that there is NO Cargo Limit and it is also Reversible Genomes containing an inserted piggyBac vector can be transiently re transfected with the PB transposase expression vector The PB transposase will remove the transposons from the genome footprint free PiggyBac PiggyBac Transposon Transposase Dp Cut Paste Chromosomal DNA at TTAA sites u H Re excise Transposase Footprint free removal 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual NOTE Grow your construct in LB Carbenicillin or 50 ug ml Ampicillin overnight shaking at 30 C using Stbl2 or Top10 cells do NOT use DH5a This is important to avoid potential undesired piggyBac vector recombination events B PiggyBac transpositions Co transfect the Super PiggyBac transposase with your PiggyBac transposon vector for integration and then use the Excision only PiggyBac transposase for footprint free transposon removal Integration PB Transposase The Super PiggyBac transposase expression vector features a st
14. r 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 2014 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 19
15. rong can CMV promoter to provide romoter robust expression of the transposase The PiggyBac LT transposase coding Naa TAi sequence has been Super PiggyBac optimized for high eee expression stability and integration activity in mammalian cells Super PiggyBac Transposase Expression Vector 5964 bp cat PB210PA 1 cane Page 4 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx Excision PB Transposase By The Excision only PiggyBac transposase expression vector features a strong CMV promoter to provide robust expression Excision Only PiggyBac 2 of the transposase The oo Promoter engineered PiggyBac secret rs Excision only transposase coding sequence has been optimized for transposon cat PB220PA 1 Pee excision only activity and f no transposon integration Excision only Ea li PiggyBacTransposase activity in mammalian cells NOTE PB210PA 1 and PB220PA 1 plasmids are designed for single use and cannot be propagated in bacteria Protocol for Piggybac Integration Into Target Cells 1 Clone the desired cDNA microRNA or shRNA into the appropriate PB vector sequence verify your clones Grow target cells to 60 80 confluency For one well of a 6 well dish 1 x 10 6 cells Combine 0 5 ug PB Transposon vector clone ex PB511B 1 0 2 ug PiggyBac Transposase vector PB210PA 1 8 0ul SBI s PureFection transfection reagent 50 ul of serum free DMEM 4 Vort
16. the number of samples Aliquot master mixes in each well in 96 well plate Add 1uL of lysate and mix by pipetting up and down Transfer 10uL of lysate SYBR mix into duplicate wells in a 384 well plate 5 Place seal cover and briefly spin to collect all the liquid 6 Use standard qPCR programs PoP 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual G Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http Awww systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Rd Mountain View CA 94043 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 Page 18 ver 13 09042014 www systembio com PiggyBac Transposon System Cat PBxxx Licensing and Warranty Statement Limited Use License Use of the PiggyBac Transposon Vector System 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
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