Neuronal Transdifferentiation Factor User Manual
Contents
1. 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual VI References Wernig M et al Direct Conversion of fibroblasts to functional neurons by defined factors Nature 463 1035 1041 2010 Vil Licensing and Warranty Use of the Mouse Neuronal Differentiation Factor Expression Lentivectors 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 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 i2. The Product containing WPRE is being transferred to you in furtherance of and reliance on such license Any use of WPRE outside of SBI s Product or the Product s intended use requires a license from the Salk Institute for Biological Studies This license agreement is effective until terminated You may terminate it at any time by destroying all Products containing WPRE in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all Products containing WPRE in you control and so notify SBI in writing This License shall be governed in its interpretation and enforcement by the laws of California Contact for WPRE Licensing The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla CA 92037 Attn Office for Technology Management Phone 858 435 4100 extension 1275 Fax 858 450 0509 Wernig s Neuron specific Transcription Factors SBI has a license from Stanford University to sell Wernig s transcription factors that are covered under US patent application 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual Serial Number 61 336 309 filed January 21 2010 any foreign patent application corresponding thereto and any divisional continuation or reexamination application and each patent that issues from any of these paten
3. fresh pre warmed MEF medium and incubate overnight The next day aspirate MEF media wash the cells with pre warmed N2B27 media twice composition can be found in Appendix B and add 2 mL of fresh pre warmed N2B27 media Incubate overnight Refresh the N2B27 media every other day until neuronal cells are observed observation may be difficult in high cell density Splitting the cells is not recommended Cells can be stained for a neuronal marker such as MAP2 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual lll Validation Data A Sequence Validation Data All constructs have been sequence verified B mRNA Expression Data Shown below are the mRNA expression validation data of the five differentiation factors and GAPD as internal control 293T and MEF cells were infected with a five factor virus pool and collected 3 days after infection for RNA extraction PCR was then performed using cDNA generated from the normalized RNA extract using gene specific primers MEF MEF 5F 293T 293T 5F mAscl1 mBrn2 mMyt1l mOlig2 mZic1 GAPD Page 8 ver 1 081211 www systembio com Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx C Functional Validation Data In the images below MEF induced neuronal cells display complex neuronal morphology left and express Tuj1 right 12 days after infection with five transcription factors IV The Re
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5. and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving Materials to be decontaminated outside of the immediate laboratory area are to be placed in a durable leakproof properly marked biohazard infectious waste container and sealed for transportation from the laboratory e Please keep in mind that pCDH vectors are integrated into genomic DNA and could have a risk of insertional mutagenesis 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual ll Protocols A Necessary Materials A midiprep or maxiprep kit Competent E coli cells pPACK packaging plasmid mix cat LV500A 1 Transfection reagent such as SBI s PureFection cat LV750A 1 e Infection reagent such as SBI s TransDux cat LV850A 1 e A virus titering kit B Packaging the lentiviruses Package the lentiviruses following SBI s guide Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells SBI offers a complete line of tools for efficient virus packaging concentration titering and subsequent transduction see Related Products in Section Ill Below is an outline of this procedure Please reference the appropriate manuals for detailed steps 1 Transform individual plasmid DNA into competent E coli cells such as Invitrogen s Stbl2 or Stbl3 using standard transformation procedure 2 Performing a midipr
6. SSBI System Biosciences Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx User Manual Store at 20 C 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 081211 contained in this user manual Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx Contents I Introduction and Background 0 A Overview eee B Expression Lentivectors offered by SBI C Safety Guidelines cee I Protocols seenen Necessary Materials ccccceceeeeeseeeeee Packaging the lentivirUS S 0 ceeseeeees D Induction Protocol c cceeeeeeeeeees Ul Validation Data cccceceeeeeteeeeeees Sequence Validation Data cccceeee mRNA Expression Data cceseeereeeees E Functional Validation Data IV Related Products n s Vz Append ocssenncseniae na Map and Features ssesseessreeeerreserresererneeres F Composing N2B27 medium G Technical Support Vi REPEFENCES eeeeeeeeeeceeeeeeeteeeeeeeeeeeeeees VII Licensing and Warranty cee 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Introduction and Background A Overview SBI offers lentiviral expression vectors of five mouse neuronal d
7. ar translocation and integration of transduced viral genome CMV Human cytomegalovirus CMV constitutive promoter for transcription Kozak Allows ribosomes to recognize the initiator codon sequence Page 10 ver 1 081211 www systembio com Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx WPRE Posttranscriptional regulatory element which enhances the stability of the viral transcripts 3 ALTR Required for viral reverse transcription self AU3 inactivating 3 LTR with deletion in U3 region prevents formation of replication competent viral particles after integration into genomic DNA SV40 Poly A_ Transcription termination and polyadenylation SV40 Ori Allows for episomal replication of plasmid in eukaryotic cells pUC ORI C Allows for high copy replication in E col AmpR C Ampicillin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand B Composing N2B27 medium Add 0 5 ml of N2 supplement to 49 5 ml of DMEM F12 medium Add 1 ml of B27 supplement to 49 ml of Neurobasal medium Mix together and filter using a 0 22um filter Store at 4 C for up to 2 weeks C Technical Support For additional information or technical assistance please call or email us at System Biosciences SBI 265 N Whisman Rd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax
8. ep or maxiprep of the lentivectors Propagate E coli cells at 30 C to reduce the chance of recombination if competent cells such as Top 10 and OmniMax were used for transformation 3 After sufficient DNA has been harvested transfect plasmid DNA individually in producing cell line with SBs pPACK which contains the necessary proteins to complete the packaging of the lentiviral particles and a transfection reagent such as SBI s PureFection 4 Harvest and concentrate the lentiviral particles from the transfection media after 48 72 hours This is easily done with SBI s PEG it solution cat LV810A 1 5 Determine the IFU mL of the viruses using a titering kit such as SBI s Global UltraRapid Lentivirual Titer Kit cat LV961A 1 Page 6 ver 1 081211 www systembio com Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx C Induction Protocol The following protocol has been optimized for MEF cells according to the method described in Wernig et al 1 w In a 6 well plate 9 6 cm well seed 1 2e5 MEF cells per well and incubate overnight in MEF media DMEM 10 FBS The next day aspirate MEF medium and add 2 mL of pre warmed MEF media and incubate for two hours Infect the MEF cells with the lentivirus pool containing 5 transcription factors each at MOI of 10 supplemented with 4ul of TransDux cat LV850A 1 The next day repeat step 2 The next day aspirate MEF media and add 2 mL of
9. he SBI website at http www systembio com stem cell research transdifferentiation factors ordering 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual C Safety Guidelines SBI s expression lentivectors together with the pPACK packaging plasmids comprise the third generation lentiviral expression system The HIV based lentivectors are based on the vectors developed for gene therapy applications by Dr J G Sodroski U S patents 5 665 577 and 5 981 276 HIV based lentivector systems are designed to maximize their biosafety features which include A deletion in the enhancer of the U3 region of ALTR ensures self inactivation of the lentiviral construct after transduction and integration into genomic DNA of the target cells The RSV promoter in HlV based vectors and CMV promoter in FIV based vectors upstream of 5 LTR in the lentivector allow efficient Tat independent production of viral RNA reducing the number of genes from HIV 1 that is used in this system Number of lentiviral genes necessary for packaging replication and transduction is reduced to three gag pol rev and the corresponding proteins are expressed from different plasmids for HIV based packaging plasmids lacking packaging signals and share no significant homology to any of the expression lentivectors pVSV G expression vector or any other vector to prevent generation of recombinant replication competen
10. ifferentiation factors Brn2 Myt1l Zic1 Olig2 and Ascl1 These five factors have been successfully used in reprogramming both mouse embryonic fibroblasts MEFs and tail fibroblasts directly to neurons iN Wernig et_al These 5 factor iNs expressed neuronal markers MAP2 NeuN and synapsin and demonstrated functional membrane properties similar to neurons Cells were positive for vGLUT 1 and GABA formed functional synapses were able to produce action potentials and contained functional voltage dependent and ligand gated ion channels Generation of iN in this fashion has an advantage of avoiding tumorigenic pluripotent cells and may provide a platform for more efficient disease modeling and drug discovery Page 2 ver 1 081211 www systembio com Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx B Expression Lentivectors offered by SBI The five mouse neuron differentiation factors are cloned into SBI s CD500B 1 HIV based expression lentivector Appendix A Expression is driven by the CMV promoter and the transcript is stabilized by the WPRE element The vector contains AmpR for selection in E colicells The neuron differentiation factors in lentivector are available individually and collectively as a set of five or as a set of three that was shown to be sufficient and most effective in conversion by Wernig et al For a full listing of all of the neuron differentiation factors available please see t
11. lated Products following related products are available at www systembio com Producer Cell Line SBI s 293 TN cell line produces high titer lentivirus Virus Packaging Systems pPACK packaging plasmid mix for optimized for lentivirus production Transfection reagent PureFection transfers DNA and siRNAs to cels with high efficiency using a nanotechnology based reagent Virus concentration Easily concentrate lentiviruses with PEG it virus precipitation solution Virus Transduction Easily transduce your target cells with TransDux Virus titering Accurate and easy qPCR measurement of lentiviral titers with the Global UltraRapid Lentiviral Titer Kit 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual V Appendix A Map and Features Map of the expression lentivectors Neuron differentiation factors mAscl1 mBrn2 mMyt1l mOlig2 and mZic1 are in the position indicated as iN Factor RSV 5 LTR cPPT CMV lt 2 Kozak iN Factor 3 ALTR WPRE The table below lists the features and functions of the lentiviral constructs Feature Function RSV 5 LTR Hybrid RSV promoter R U5 long terminal repeat required for viral packaging and transcription gag Packaging signal RRe Rev response element binds gag and involved in packaging of viral transcripts cPPT Central polypurine tract includes DNA Flap region involved in nucle
12. s strictly prohibited without written permission by System Biosciences HIV Vector System This product is for non clinical research use only Use of this Product to produce products for resale or for any diagnostic therapeutic clinical veterinary or food purpose is prohibited In order to obtain a license to use this Product for these commercial purposes contact the Office of Research and Technology Ventures at the Dana Farber Cancer Institute Inc in Boston Massachusetts USA This Product or the use of this Product is covered by U S Patents Nos 5 665 577 and 5 981 276 owned by the Dana Farber Cancer Institute Inc Page 12 ver 1 081211 www systembio com Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx WPRE Technology System Biosciences SBI has a license to sell the Product containing WPRE under the terms described below Any use of the WPRE outside of SBI s Product or the Products intended use requires a license as detailed below Before using the Product containing WPRE please read the following license agreement If you do not agree to be bound by its terms contact SBI within 10 days for authorization to return the unused Product containing WPRE and to receive a full credit The WPRE technology is covered by patents issued to The Salk Institute for Biological Studies SBI grants you a non exclusive license to use the enclosed Product containing WPRE in its entirety for its intended use
13. t applications CMV Promoter The CMV promoter is covered under U S Patents 5 168 062 and 5 385 839 and its use is permitted for research purposes only Any other use of the CMV promoter requires a license from the University of lowa Research Foundation 214 Technology Innovation Center lowa City IA 52242 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
14. t virus None of the HIV 1 genes gag pol rev will be present in the packaged viral genome as they are expressed from packaging plasmids lacking packaging signal therefore the lentiviral particles generated are replication incompetent Pseudoviral particles will carry only a copy of your expression construct Despite the above safety features use of SBI s lentivectors falls within NIH Biosafety Level 2 criteria due to the potential biohazard Page 4 ver 1 081211 www systembio com Mouse Neuronal Differentiation Factor Expression Lentivectors Cat TD2xxx risk of possible recombination with endogenous viral sequences to form self replicating virus or the possibility of insertional mutagenesis For a description of laboratory biosafety level criteria consult the Centers for Disease Control Office of Health and Safety Web site at http www cdc gov od ohs biosfty bmbl4 bmbl4s3 htm It is also important to check with the health and safety guidelines at your institution regarding the use of lentiviruses and always follow standard microbiological practices which include e Wear gloves and lab coat all the time when conducting the procedure e Always work with pseudoviral particles in a Class Il laminar flow hood e All procedures are performed carefully to minimize the creation of splashes or aerosols e Work surfaces are decontaminated at least once a day and after any spill of viable material e All cultures stocks
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