Lenti-miR™ Virus Library
Contents
1. L e Recover genomic DNA 5 LVL Primer 3 LVL Primer Amplify microRNA clone Directly sequence M 1 2 3 4 5 6 gi Ss bp 1000 500 PCR to recover the exogenous pre microRNA sequences Page 4 ver 1 2008 06 10 www systembio com Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 Protocol General considerations The transduction efficiency of the Lenti miR library varies significantly for different cells and experimental conditions In order to optimize transduction conditions we recommend that you use the positive control CD511VB 1 virus provided in this kit to determine the optimal MOI for your cells of interest see below Determining the Optimal MOI MOI is defined as the number of transducing units per cell A number of factors can influence determination of an optimal MOI including the nature of your mammalian cell actively versus non dividing its transduction efficiency your application of interest If you are transducing the Lenti miR virus library into the your cells for the first time we recommend using a range of MOls e g 1 2 5 10 to determine the MOI required to obtain optimal expression for your particular application Efficiency of transduction of your cell type with the positive control virus CD511VB 1 or the Lenti miR virus library can be monitored by copGFP expression Expression of the microRNA precursors can be measured directly at2. SBI System Biosciences Lenti miR Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 User Manual Store kit at 80 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 ver 1 2008 06 10 contained in this user manual Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 Contents l Introduction and Background A Purpose ofthisManual sss 2 B MicroRNA Background usse 2 C Product Description ss 3 D Intended Use 4 ll Protocol A General Considerations sss 5 B Transduction of Lenti miR Virus Library sss sss 6 C Phenotypic Selection ss 7 D Recovery of MicroRNA Effector Clone Sequences 7 IIl Troubleshooting 10 IV Appendix A Lenti miR microRNA precursor constructs 12 B Production and Titering of Lenti miR Virus Library 14 C ListofKitComponents Less 15 D Related Products sn 15 E Technical Support sss sss 16 F Safety Guidelines sss 16 V References 18 VI Licensing and Warranty Statement 19 This Product shall be used by the purchaser for internal research purposes only and distribution is strictly prohibited without written permission by System Biosciences 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual l Introduction and Background A Purpose of t
3. ver 1 2008 06 10 www systembio com Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 C Phenotypic Selection Day 4 to 6 By 48 to 72 hours post transduction copGFP expression will be apparent in infected cells e Transient transduction screen Begin your phenotypic analysis and select cells exibiting the desired phenotypes e Stable transduction screen Begin your FACS sort for copGFP positive cells to enrich for stably transduced cells Establish a stably transduced cell population before performing your phenotypic analysis D Recovery of Effector MicroRNA Clone Sequences Materials required and not supplied in kit For Purification of genomic DNA DNeasy Tissue Kit QIAGEN Cat 69504 or equivalent For PCR Amplification Standard PCR kit TITANIUM Tag PCR Kit Clontech Cat 639210 or equivalent Thermal Cycler DNA Engine MJ Research Cat PTC 200 or equivalent 2 5 1X TAE Agarose gel 1 Purify Genomic DNA from Selected Cells from Lenti miR Phenotypic Screen Prepare genomic DNA from selected cells according to manufacturer s recommendations Measure the yield of DNA by spectrophotometer You should expect to isolate 5 10 ug of genomic DNA from approximately 1x10 cells Dilute the DNA sample in deionized water to a concentration of 0 2 ug l 2 An sample PCR reaction for microRNA effector clone sequence amplification is provided below A Prepare a PCR Master Mix for all reactio
4. Notl sites of the pMIRNAfT lentivector The HIV 1 derived pMIRNA1 pCDH vectors contain the following common features WPRE element enhances stability and translation of the CMV driven transcripts e SV40 polyadenylation signal enables efficient termination of transcription and processing of recombinant transcripts Hybrid RSV 5 LTR promoter provides a high level of expression of the full length viral transcript in producer 293 cells e Genetic elements cPPT GAG LTRs necessary for packaging transducing and stably integrating the viral expression construct into genomic DNA e SV40 origin for stable propagation of the pMIRNA1 plasmid in mammalian cells e pUC origin for high copy replication and maintenance of the plasmid in E coli cells Ampicillin resistance gene for selection in E coli cells e copGFP fluorescent marker to monitor cells positive for transfection and transduction Properties of the copGFP Fluorescent Protein The pMIRNA1 Vector contains the full length copGFP gene with optimized human codons for high level of expression of the fluorescent protein from the CMV promoter in mammalian cells The copGFP marker is a novel natural green monomeric GFP like protein from copepod Pontellina sp The copGFP protein is a non toxic non aggregating protein with fast protein maturation high stability at a wide range of pH pH 4 12 and does not require any additional cofactors or su
5. about 48 72 hours after transduction For more information refer to the QuantiMir small RNA quantitation system s User manual www systembio com Transient versus Stable screen Depending on the nature of your phenotypic screen you may use the Lenti miR virus library to transiently or stably transduce your cells Tranduced cells can be sorted by flow cytometry based upon copGFP expression Biosafety considerations Although these viral particles are replication incompetent they can infect mammalian cells and integrate into the host cell genome For detailed biosafety information see Appendix F Please follow all relevant Federal State and Local regulations for working with BSL 2 class viruses 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual B Transduction of Lenti miR Virus Library The following protocol provides procedures for transduction of the Lenti miR virus library into target cells and is optimized for adherent cell lines such as HeLa or MCF7 in a 6 well plate format Use these guidelines as a starting point for determining optimal conditions for your cells and experiments To use a different culture dish adjust the number of cells volumes of reagents and quantities of virus in proportion to the surface area of the dish well see Table below a eT Tissue Culture Dish per well cm2 volume per wer voume o u o 9mm 8 2 PG well So 94 S 2 EET CNN NN M
6. effective means to concentrate lentiviral vector inocula produced with SBI s pPACK Lentivector packaging systems LentiBrene Cat LV811A 1 Lentivector UltraRapid Titer PCR Kit Cat LV960A 1 for human cells LV960B 1 for mouse cells Allows you to measure copy number MOI of integrated lentiviral constructs in genomic DNA of target cells after transduction with constructs made in any of SBI s FIV or HIV based Lentivectors or GeneNet siRNA Libraries QuantiMir small RNA quantitation system Cat RA420A 1 Easily profile miRNAs using a single cDNA synthesis step and real time qPCR MicroRNA Precursor Clone collection cat s pMIRHxxxPA 1 SBI s collection of individual microRNA precursor expression clones in lentivectors 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual E 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 System Biosciences SBI 1616 North Shoreline Blvd 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 2systembio com Ordering Information orders systembio com Safety Guidelines SBI s Expression lentivectors together with the pPA
7. vectors Replication incompetent HIV based vectors are considered biologically safe RNA Polymerase ll promoter ensures robust expression from single copy integrants While pol Ill promoters are required to express very short siRNA and shRNA the primary microRNAs can be expressed using conventional pol Il promoters Stegmeier 2005 The traditionally utilized pol lll promoters H1 and U6 are constitutively expressed in all cell types which complicates studies where knockdown of a gene product is lethal Different pol Il promoters can alternatively provide either spatially or temporally defined expression Shin 2006 SBI s microRNA Precursor Clone Collection expresses each miRNA precursor from the CMV promoter This robust strong viral promoter ensures a high level of expression from a single copy of integrated miRNA construct 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual Dual expression design simplifies identification of transduced cells The unique organization of the vector below results in expression of a another downstream transcript containing the copGFP fluorescent marker driven by the Human EF 10 promoter RSV 5 LTR AmpR j as pMIRNA1 pUC ORI 7 544 bp microRNA SV40 ORI WE cr Precursor WPRE COPGFP Map of HIV based lentiviral plasmid expressing one of the microRNA precursors from SBI s collection All MicroRNA Precursors are cloned into the EcoRI
8. 1 PMIRHPLVAHT 1 IV APPENDIX A Lenti miR MicroRNA Precursor Constructs System Biosciences SBI s microRNA Precursor Construct Collection incorporates several unique features that set it apart from any commercially available miRNA collection Express microRNA precursor transcripts from their native genomic context The inserts in SBI s microRNA Precursor Construct Collection represent more than just the mature microRNA sequences listed in Sanger s miRBase http microrna sanger ac uk sequences Each construct in SBI s collection consists of the stem loop structure and 300 500 base pairs of upstream and downstream flanking genomic sequence This unique feature ensures that the microRNAs expressed from SBI s clones act as naturally as possible The native structure ensures interaction with endogenous RNA processing machinery and regulatory partners leading to properly cleaved microRNAs Lentiviral transduction system is effective and safe Each of SBI s miRNA precursor molecules has been cloned in a lentiviral based vector Like other standard plasmid vectors SBI s miRNA construct can be used for transient expression of miRNA using conventional transfection protocols Moreover its lentiviral backbone allows each miRNA construct to be packaged in pseudoviral particles and introduced into non dividing or difficult to transfect cell lines In particular all of the miRNAs have been cloned in SBI s HIV based expression
9. CK 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 US patent 5 665 577 and 5 981 276 Both FIV based and HIV based lentivector systems are designed to maximize their biosafety features which include e A deletion in the enhancer of the U3 region of S ALTR ensures self inactivation of the lentiviral construct after transduction and integration into genomic DNA of the target cells e The RSV promoter in HIV based vectors and CMV promoter in FlV 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 are used in this system e 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 competent virus e 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 e Pseudovi
10. Day 1 1 Plate target cells in a 6 well plate at a density of 1x10 cells per well 24 hours prior to viral infection in 2 ml of complete medium with serum and antibiotics Incubate cells at 37 C with 5 CO overnight Typically cells will be 30 5096 confluent at the time of infection Day 2 2 Dilute an appropriate amount depending on the optimal MOI of virus into 0 5 ml of complete medium Add Polybrene example LentiBrene SBI cat LV811A 1 to a final concentration of 5 8 ug ml IN THIS EXAMPLE Q 100 000 cells well were seeded on Day 1 O Assume doubling overnight yields 200 000 cells well on Day 2 Q To infect at an MOI of 5 use 1 x 10 IFUs virus per well Note Polybrene is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cellular membrane The optimal concentration of Polybrene depends on cell type and may need to be empirically determined usually in the range of 2 10 ug ml Excessive exposure to Polybrene gt 12 hr can be toxic to some cells Remove the culture medium from cells Infect target cells by adding the viral stock dilutions to the wells For one well mock well control add 0 5 ml of D MEM medium with Polybrene Incubate cells at 37 C with 5 CO overnight Day 3 4 Remove the culture medium and replace with 2 ml of complete medium without Polybrene Incubate the cells at 37 C with 5 CO2 overnight Page 6
11. RNA precursor sequence responsible for the observed phenotypes 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual D Intended use The Lenti miR virus library is a tool that enables the study of phenotypic effects associated with the over expression of individual microRNAs The lentivirus preparation is pseudotyped with VSV G that allows for broad cellular tropism Hard to transfect mammalian cell lines primary cells non dividing cells and even whole animal studies are possible Transduced cells exhibiting the phenotypes of interest are isolated by selection or sorting The microRNA or microRNAs responsible for generating the phenotypes of interest may be recovered through simple genomic PCR using lentivector specific primers followed by direct sequencing of microRNA precursor clones FIG 2 An overall 1 tM Vi z Lenti miR Virus Library experimental workflow for Phenotypic Screens virus transduction phenotypic selection is Simultaneously over express SBI s full complement of Human MicroRNAs i shown at left Schematic 5 for the recovery of the e MicroRNA effectors is Infect 1 shown at bottom e Cell lines e Stem cells e Animal tissues Select tL cells exibiting phenotype Tumor Invasion MEAT SSS Identify MicroRNA Effectors e Senescence e Apoptosis l CMV copGFP Identify MicroRNAs MicroRNA effectors ll gt Isolate desired cells
12. Y Liu J V Duijse J Drost A Griekspoor E Zlotorynski N Yabuta G D Vita H Nojima L H J Looijenga and R Agami 2006 A Genetic Screen Implicates miRNA 372 and miRNA 373 As Oncogenes in Testicular Germ Cell Tumors Cell 124 1169 1181 Olsen P H and V Ambros 1999 The lin 4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN 14 protein synthesis after the initiation of translation Dev Biol 216 2 671 80 Pfeffer S M Zavolan F A Grasser M Chien J J Russo J Ju B John A J Enright D Marks C Sander and T Tuschl 2004 Identification of virus encoded microRNAs Science 304 5671 734 6 Poeschla E M Looney D J and Wong Staal F 2003 Lentiviral nucleic acids and uses thereof US Patent NO 6 555 107 B2 Shin K J E A Wall J R Zavzavadjian L A Santat J Liu J Hwang R Rebres T Roach W Seaman M Simon and I D Fraser 2006 A single lentiviral vector platform for microRNA based conditional RNA interference and coordinated transgene expression Proc Natl Acad Sci U S A 103 37 13759 64 Stegmeier F G Hu R J Rickles G J Hannon and S J Elledge 2005 A lentiviral microRNA based system for single copy polymerase II regulated RNA interference in mammalian cells Proc Natl Acad Sci U S A 102 37 13212 7 Yi R Y Qin G Macara and B R Cullen 2003 Exportin 5 mediates the nuclear export of pre microRNAs and
13. bstrates The copGFP protein has very bright fluorescence that exceeds at least 1 3 times the brightness of EGFP the widely used Aequorea victoria GFP mutant The copGFP protein emits green fluorescence with the following characteristics emission wavelength max 502 nm Page 12 ver 1 2008 06 10 www systembio com Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 excitation wavelength max 482 nm quantum yield 0 6 extinction coefficient 70 000 M cm Due to its exceptional properties copGFP is an excellent fluorescent marker which can be used instead of EGFP for monitoring delivery of lentivector constructs into cells 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual B Production and Titering of Lenti miR Virus Library The Lenti miR Virus Library was prepared as follows The full set of SBI s human precursor microRNA expression clones was pooled and packaged into VSV G pseudotyped viral particles by co transfecting 293TN Producer Cells SBI Cat LV900A 1 with the library clones and pPACKH1 SBI Cat LV500A 1 according to the procedures described in the Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells Virus was concentrated with PEG it Virus Precipitation Solution SBI Cat LV810A 1 according to the instructions in the User Manual Infectious titers were determined on HT1080 cells using the UltraRapid Le
14. c 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 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 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
15. his Manual This manual provides information about the design and production of the Lenti miR virus library and detailed instructions on its use The Lenti miR virus library contains all of SBI s microRNA precursor clones as a pooled ready to infect VSV G pseudotyped lentiviral preparation For more detailed information regarding the individual clones that comprise the library please refer to the user manual for the PMIRHxxxPA 1 product line this is available online at www systembio com Before using this reagent and associated materials provided in this kit please read the entire manual B MicroRNA Background The term microRNA miRNA describes a novel class of small non coding RNAs which regulate gene expression post transcriptionally by disrupting translation or directing cleavage of complementary mRNAs These 17 26 nucleotide nt single stranded miRNA molecules are synthesized as primary transcripts pri miRNA that are often polycistronic containing a small number of clustered miRNA units Following transcription and while the transcript still remains nuclear the Drosha RNAse III nuclease processes the pri miRNA into 70nt stem loop precursors pre miRNA These pre miRNA molecules are transported to the cytoplasm by a complex of proteins which includes the dsRNA binding protein Exportin 5 where they are processed to their final mature form by another RNAse III nuclease Dicer Lee 2002 Yi 2003 It is here in the cy
16. icroRNA sequences CMV Pre microRNA Pre microRNA clone Primer x f MicroRNAs M 155 195 221 222 224 30e 34a 373 _ Detection of exogenous pre microRNAs after infection with the pre microRNA collection A Primers used for amplification B PCR results Note that a unique band for each of microRNA as indicated by a yellow arrow M DNA markers 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Ill Troubleshooting 1 Poor Infection Efficiency Too high a volume of infecting supernatant Keep the volume as low as possible to achieve maximal adsorption of viral particles to the cells The assay is carried out too early Normally the maximal expression of integrated provirus is expected to develop by 48 hours after infection However some cells display delayed expression Try the assay at a later time such as 72 or 96 hours Target cell line may be difficult to transduce Optimize the transduction protocol Use a higher MOI Wrong amount of Polybrene added during titration Add and optimize Polybrene concentration in the range of 4 10 ug ml Loss of pseudoviral titer during storage Store pseudoviral stock at 80 C Each freeze thaw cycle drops the titer 2 4 fold Use a fresh aliquot for transduction Infection Affects Target Cell Viability High MOI may be toxic to some cell types Reduce the amounts of virus used Polybrene is toxic for target cells Optimize
17. iding 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 2008 System Biosciences SBI Page 20 ver 1 2008 06 10 www systembio com
18. n tubes plus one additional tube Combine the following components in the order shown 4i y Deionized H2O 5 oul 10X PCR buffer 1 ou 50X dNTP mix 10 mM of each dNTP 1 ou LVL Primer Mix 10 uM of each primer 1 ou Purified Genomic DNA 200ng 1 ou 50X Taq DNA polymerase 50 ul Total volume Note Include no DNA negative control B Mix contents by vortexing and spin the tube briefly in a microcentrifuge C Perform PCR amplification according to these guidelines 94 C for 2 min 94 C for 30 sec 60 C for 30 sec for 25 cycles 68 C for 1 min 15 C hold D When the program is completed analyze a 10 ul sample from each tube and suitable DNA size marker 200 bp 2 kb on a 1 596 agarose EtBr gel in 1X TAE Note that each microRNA precursor clone within the library collection varies in length 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Page 8 Therefore the expected amplicon size may range from 500 to 600 bp see sample data on the following page Sequence the effector microRNA amplicons recovered after gel purification using either the Forward or Reverse PCR primer PCR primer sequences are provided in Appendix C ver 1 2008 06 10 www systembio com Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 An example of a successful microRNA effector sequence amplification is shown below Primer 1 To recover the exogenous pre m
19. ntiviral Titering Kit for Human Cells SBI Cat LV960A 1 according to the recommended procedures Additional information and User Manuals for each of these products are available on our website www systembio com Sample Transduction Data Easily monitor infection efficiency by GFP expression Bright Field GFP P m onm E GFP expression in HT1080 cells transduced with the Lenti miR Virus Library Page 14 ver 1 2008 06 10 www systembio com C Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 List of Kit Components Aliquots of pooled Lenti miR virus library Q Tube of positve infection control virus CD511VB 1 Q Tube of LVL PCR Primer Mix 10 uM of each primer o pCDH 5 1 5 GCC TGG AGA CGC CAT CCA CGC TG 3 o pCDH 3 1 5 GAT GTG CGC TCT GCC CAC TGAC 3 Related Products pPACKH1 Lentivector Packaging Kit Cat LV500A 1 Unique lentiviral vectors that produce all the necessary HIV viral proteins and the VSV G envelope glycoprotein from vesicular stomatitis virus required to make active pseudoviral particles 293TN cells SBI Cat LV900A 1 transiently transfected with the pPACKH1 and a pMIRNA1 microRNA expression construct produce packaged viral particles containing a microRNA construct 293TN Human Kidney Producer Cell Line SBI Cat LV900A 1 For packaging of plasmid lentivector constructs PEG it Virus Precipitation Solution Cat LV810A 1 Simple and highly
20. 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 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 CopGFP Reporter This product contains a proprietary nucleic acid coding for a proprietary fluorescent protein s intended to be used for research purposes only Any use of the proprietary nucleic acids other than for research use is strictly prohibited USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN To obtain such a license please contact Evrogen at license evrogen com 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual SBI has pending patent applications on various features and components of the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any
21. ral 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 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 ombl4 ombl4s3 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 Page 16 ver 1 2008 06 10 www systembio com Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 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 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 leak proof properly marked biohaza
22. rd infectious waste container and sealed for transportation from the laboratory e Please keep in mind that pMIRNA1 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 17 System Biosciences SBI User Manual V References Bennasser Y S Y Le M L Yeung and K T Jeang 2004 HIV 1 encoded candidate micro RNAs and their cellular targets Retrovirology 1 1 43 Hariharan M V Scaria B Pillai and S K Brahmachari 2005 Targets for human encoded microRNAs in HIV genes Biochem Biophys Res Commun 337 4 1214 8 John B C Sander and D S Marks 2006 Prediction of human microRNA targets Methods Mol Biol 342 101 13 Johnson S M H Grosshans J Shingara M Byrom R Jarvis A Cheng E Labourier K L Reinert D Brown and F J Slack 2005 RAS is regulated by the let 7 microRNA family Cell 120 5 635 47 Kim V N 2005 Small RNAs classification biogenesis and function Mol Cells 19 1 1 15 Lee R C R L Feinbaum and V Ambros 1993 The C elegans heterochronic gene lin 4 encodes small RNAs with antisense complementarity to lin 14 Cell 75 5 843 54 Lee Y K Jeon J T Lee S Kim and V N Kim 2002 MicroRNA maturation stepwise processing and subcellular localization Embo J 21 17 4663 70 Mathijs Voorhoeve P C L Sage M Schrier A J M Gillis H Stoop R Nagel
23. 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 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 prov
24. short hairpin RNAs Genes Dev 17 24 3011 6 Page 18 ver 1 2008 06 10 www systembio com Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 Vi Licensing and Warranty Statement Limited Use License Use of the Lenti miR virus library 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 HIV Vector System This Product or the use of this Product is covered by U S Patents Nos 5 665 577 and 5 981 276 and foreign equivalents owned by the Dana Farber Cancer Institute Inc and licensed by SBI This product is for non clinical research use only Use of this Product to produce products for resale or for any diagnosti
25. the concentration and exposure time to Polybrene during the transduction step No Expression of microRNA precursors The CMV promoter is not functional in target cells It is a very rare case but the only way to solve this problem is to change the type of target cells No PCR product Amplified The amount of genomic DNA in your sample is too low Repeat purification of genomic DNA from infected cells or measure the concentration of genomic DNA again using a smaller dilution factor Sequence of microRNA effector is ambiguous If direct sequencing of the recovered and PCR amplified microRNA effector does not produce a single sequence your screen may have identified two or more co effector microRNAs that may contribute to your phenotype Your cells may have been transduced by more than one virus The observed phenotype may be due to expression of one or more microRNAs To identify and separate the individual microRNA effectors A Clone the amplicon s into a suitable PCR cloning vector TA or TOPO plasmids Invitrogen B Sequence at least 10 individual clones to identify candidate effector microRNAs C Test individual candidates for the phenotype of interest If no individual microRNA reproduces the observed phenotype test combinations of the candidates identified above All individual microRNA precursor clones are available from SBI Page 10 ver 1 2008 06 10 www systembio com Lenti miR Pooled Virus Library Cat s PMIRHPLVA
26. toplasm that mature miRNAs ultimately affect the protein levels of their target mRNAs by binding to complementary regions and either inhibiting translation or directing mRNA cleavage Kim 2005 see FIG 1 MicroRNA precursor Drosha processing 5 3 Dicer processing dsRNA Intermediate FIG1 Processing of 5 at microRNA precursors Kan myt The Lenti miR library will express both the both the miR miR mature miR and the miR star forms 3 rrrmmIMEITEITE S 5 ULULLLLLLLLLLLLLLLLI 3 iR miR separated mature miR RISC complexes translational repression mRNA decay Page 2 ver 1 2008 06 10 www systembio com C Lenti miR Pooled Virus Library Cat s PMIRHPLVA 1 PMIRHPLVAHT 1 Product Description The Lenti miR virus library contains a pool of SBI s microRNA precursor clones in a ready to infect lentiviral preparation Each virus within the pool will express an individual miRNA precursor in its native context while preserving putative hairpin structures to ensure biologically relevant interactions with endogenous processing machinery and regulatory partners Details of the individual microRNA precursor clone construction and preparation of the virus pool are described in Appendices A and B respectively Also included in this kit are a positve control virus CD511VB 1 to assess transduction efficiency in your cells of interest and the oligonucleotide primers that may be used to identify the micro
Download Pdf Manuals
Related Search