MicroRNA Constructs Manual
Contents
1. includes DNA Flap cPPT 1798 1916 region involved in nuclear translocation and integration of transduced viral genome ha Human cytomegalovirus CMV constitutive GMN promoter i rica promoter for transcription of cloned cDNA insert Elongation factor 1a promoter constitutive EF1 2315 2860 promoter for transcription of Reporter gene Puromycin resistance or copGFP Copepod green fluorescent protein similar to copGFP 2874 3629 regular EGFP but with brighter color as a reporter for the transfected transduced cells Woodchuck hepatitis virus posttranscriptional WPRE 3639 4229 regulatory element enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR AU3 4301 4534 inactivating 3 LTR with deletion in U3 region l prevents formation of replication competent viral particles after integration into genomic DNA SV40 Poly A 4606 4737 Transcription termination and polyadenylation SV40 Ori 4746 4892 Allows for episomal replication of plasmid in eukaryotic cells pUC Ori 5262 5935 C Allows for high copy replication in E coli AmpR 6080 6940 C Ampicillin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand 650 968 2200 outside US Page 15 System Biosciences SBI User Manual C Properties of the copGFP Fluorescent Protein The pMIRNA1 Vector contains the full length copGFP gene with optimized human codons for high le2. 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 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 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 and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving Materials to be decontam
3. in its native expressed form The primary microRNA contains critical biological components involved in mature miRNA expression and cellular processing it often been processed into several mature miRNA molecules The second major drawback of synthetic miRNA molecules is that their knockdown effect in the target cell is transient and usually disappears 2 3 days after transfection SBI s microRNA Precursor constructs express each individual miRNA precursor in its native context while preserving putative hairpin structures to ensure biologically relevant interactions with endogenous processing machinery and regulatory partners The lentiviral expression system also makes it possible to have a stable knockdown effect after being introduced into the cells Page 2 ver 2 082412 www systembio com MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 Unique Features of SBI s 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 SBl s microRNA Precursor Construct Collection represent more than just the mature microRNA sequences listed in Sangers 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 downstre
4. on screens with pooled microRNA libraries please see the following references Voorhoeve PM le Sage C Schrier M Gillis AJ Stoop H Nagel R Liu YP van Duijse J Drost J Griekspoor A Zlotorynski E Yabuta N De Vita G Nojima H Looijenga LH Agami R A genetic screen implicates miRNA 372 and miRNA 373 as oncogenes in testicular germ cell tumors Cell 2006 Mar 24 124 6 1169 81 Huang Q Gumireddy K Schrier M le Sage C Nagel R Nair S Egan DA Li A Huang G Klein Szanto AJ Gimotty PA Katsaros D Coukos G Zhang L Pur E Agami R The microRNAs miR 373 and miR 520c promote tumour invasion and metastasis Nat Cell Biol 2008 Feb 10 2 202 10 Page 12 ver 2 082412 www systembio com MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 ll Appendix A Map of pMIRNA1 RSV 5 TR AmpR RRE env cPPT pMIRNA1 pUC ORI 7 544 bp CMV microRNA SV40 ORI EF1 precursor SV40 poly A 3 ALTR WPRE CopGFP To sequence your pMIRNA 1 clone EF1 rev primer 5 GCACCCGTTCAATTGCCG 3 The sequence read will be the opposite strand of the sense strand miRNA precursor 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual To verfiy the miRNA precursor in your pMIRNA1 clone Use web BLAST at Sanger miRBase http microrna sanger ac uk sequences search shtml Wat be Clio Sey taboo FER x n miRBase Sequences Select Stem loop
5. sequences Select organism chromosome and start end end coordinates Leave the stert end bexcs blank te retricve all miRNAs on thc selcctec chromosome See yens 9 Che Srl Jimi sequences Single sequence searches Paste 2 sequence here to search against MIRNA sequences choose to search against the intact precursor sequences or Juspe mature MIRNAS I his search may teke 2 tew minutes Max size 1000bp s Search sequences Ezom sop esq ate Search melhod easi Choose BLASI N to search for a MIRNA in a longer sequence SSLARCII Is useful for finding e short sequence within the library ot mIRNAs tor Instance tind a short motit In a mIRNA or precursor stem loop or find mature sequences that are related to your query Or lin the sequence file yem wish lo use Grn Ie Maximum no of hits e 0 _ vem minas ites Eregi p Mice rhet AI T STEP 1 Copy Paste your raw sequence data into the Box above marked 1 STEP 2 Selct from the drop down box the Stem loop sequences selection marked 2 above Page 14 ver 2 082412 www systembio com MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 B Features of pMIRNA1 888 266 5066 Toll Free Feature Location Function RSV S LTR 7 414 Hybrid RSV promoter R US long terminal repeat required for viral packaging and transcription gags 567 919 Packaging signal packaging of viral transcripts Central polypurine tract
6. Invitrogen Lipofectamine Cat 18324 111 and Plus Reagent Cat 11514 015 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual G 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 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 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 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 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
7. Precursor Construct 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 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 Mas
8. SSB System Biosciences Lentivector based MicroRNA Precursor Constructs Cat PMIRHxxxPA 1 User Manual Grow bacterial stock on receipt Make plasmid DNA for experiments 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 2008 02 28 001 contained in this user manual MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 Contents l Introduction and Background A Purpose of thisManual ee 2 B OVCIVIOW o a ia anene a a iam COME maaeandensead dae 2 C Tools for Functional Study of MicroRNA secu 2 D Unique Features of SBI s MicroRNA Precursor Constructs sss 3 E List of Components sess arara anan 7 F Additional Required Materials sss ee i DllDllllllll 7 G Safety Guidelines sss ssl sslDllDllDlllilDlllilDllll 8 ll Protocol A Applications of SBI s MicroRNA Precursor Constructs sus 9 B Transfection of microRNA Precursor Constructs sss uuu 10 C Packaging SBI s Cloned MicroRNA Precursor Constructs sss 11 lll References LLL 12 IV Appendix A Map of pMIRNA1 and verifying clone content sss 13 B Features of pMIRNAd ss 15 C Properties of copGFP Fluorescent Protein eee ee 16 D Related Products sss ec 16 E Technical Support sss LLL Lll 16 V Licensing and Warranty Statement sss 17 i This Product shall be used by the purchaser for internal research purposes only a
9. act 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 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 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 perso
10. after overnight incubation please contact SBI immediately for a replacement 650 968 2200 e Pick a single isolated colony from the plate and grow a liquid culture of suitable volume in LB ampicillin 100 ug ml shaking at Plasmid purification kit Recommended QIAGEN Endotoxin free Plasmid Kit The following kit combinations can be used for Midi scale up to 200 ug of plasmid DNA preparation of endotoxin free DNA gt QIAfilter Plasmid Midi Kit Cat 12243 and EndoFree Plasmid Maxi Kit Cat 12362 gt QIAfilter Plasmid Midi Kit Cat 12243 and EndoFree Plasmid Buffer Set Cat 19048 Please visit the QIAGEN website to download the specialized protocol that is not contained in the current user manual gt http www1 qiagen com literature protocols pdf QP15 pdf For Transfection of pMIRNA1 Constructs into Target Cells e Transfection Reagent Recommended Invitrogen Lipofectamine 2000 Cat 11668 027 For Packaging of pMIRNA1 Constructs in Pseudoviral Particles e In order to package your pMIRNA1 construct into VSV G pseudotyped viral particles you will need to purchase the pPACKH 1 Lentivector Packaging Kit Cat LV500A 1 The protocol for packaging and transduction of packaged pseudoviral particles is provided in the User Manual for the Lentivector Expression System e 293 Producer Cell Line Recommended SBI 293TN Cell Line Cat LV900A 1 or ATCC 293 Cells Cat CRL 11268 e Transfection Reagent Recommended
11. am 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 MicroRNA precursor Drosha processing Dicer processing dsRNA Intermediate Ny m 31 5 miR miR 4 mimm 5l 5 LLLLLLLELLLLLLLLLLLIE 37 iR miR separated nature mig RISC complexes translational repression mRNA decay MicroRNA Precursors expressed from SBI s Lentivectors produce Mature microRNAs 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual 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 vectors Replication incompetent HIV based vectors are considered biologically safe RNA Polymerase ll promoter ensures robust expression from single copy integrants While pol lll promoters are required to express v
12. cursors 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 Ill nuclease Dicer Lee 2002 Yi 2003 It is here in the cytoplasm 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 Initially discovered in C elegans as subtle regulators of cell fate miRNAs have since been identified in all metazoan organisms controlling such vital processes as cell proliferation differentiation signal transduction and cell death The lin 4 and let 7 miRNAs of the nematode were found to modulate gene expression by binding to complementary sites in the 3 UTR of the target gene s mRNA Lee 1993 Olsen 1999 This binding of microRNAs affords the cell post transcriptional control over gene expression and adds another layer to the already complex gene expression regulatory network Recently miRNAs were implicated in the biogenesis of human diseases It was shown that human lung cancer tumors had distinctly lower levels of the let 7 miRNA Johnson 2005 This is a critical observation because the RAS proto oncogene family has sites that are complementary to let 7 enabling let 7 mediated control of RAS expression RAS proteins were upregulated in these lung tumors su
13. ed into 293 cells miRNA qPCR measurements done with QuantiMir T o SAH RHA undi at on Sychesni Fig 3 High levels og miRNAs deteced from pMIRNA1 constructs Real time qPCR with the QuantiMir small RNA quantitation system was used to evaluate expression from the pMIRNA clones shown to the left QuantiMir KIt SBI cat RA420A 1 Page 6 ver 2 082412 www systembio com MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 E List of Components MicroRNA Precursor Construct Collection e The microRNA precursor constructs are shipped in bacterial stock form E coli plated on LB carbenicillin at 50ug ml same as ampicillin but more stable over time for shipment e Upon receipt individual colonies may be apparent already If no colonies are visible upon receipt incubate the LB carbenicillin overnight at 37 C If there are no apparent colonies after overnight incubation please contact SBI immediately for a replacement 650 968 2200 Example of bacterial stock streak for PreMiR 513a 1 F Additional Required Materials For Purifying Construct Plasmid DNA after Receipt e The microRNA precursor constructs are shipped in bacterial stock form E coli plated on LB carbenicillin at 50ug ml same as ampicillin but more stable over time for shipment e Upon receipt individual colonies may be apparent If no colonies are visible upon receipt incubate the LB carbenicillin overnight at 37 C If there are no apparent colonies
14. ent 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 SV40 origin for stable propagation of the pMIRNA1 plasmid in mammalian cells 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 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Easily monitor expression positive cells using GFP fluorescence GFP Fluorescence Peeks st 293TN Cells transfected with 500 ng PreMiR 513a 1 cat PMIRH5131PA 1 using Lipofectamine 2000 Invitrogen Photos taken after 48h Fig 2 Phase contrast and GFP fluoresence images of 293TN cells after transfection of 500 ng PreMiR 513a 1 construct DNA using Lipofectamine 2000 Invitrogen Robust mature miRNAs produced using SBI s MicroRNA Precursor Constructs Sample pre miRNA Clone Expression Validation Data Endogenous levels m miRNA precursor expression 1000 Relative expression level 3825 858 33235 2 Individual Pre miRNA Clones transfect
15. ery short siRNA and shRNA the primary microRNAs can be expressed using conventional pol ll promoters Stegmeier 2005 The traditionally utilized pol Ill 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 Page 4 ver 2 082412 www systembio com MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 Dual expression design simplifies identification of transduced cells The unique organization of the vector Fig 1 results in expression of a another downstream transcript containing the copGFP fluorescent marker driven by the Human EF 10 promoter see Fig 2 RSV S LTR AmpR RRE env cPPT CMV K microRNA g Precursor pMIRNA1 7 544 bp pUC ORI SV40 ORI SV40 poly A TALE WPRE copGFP Fig 1 Map of HIV based lentiviral plasmid expressing one of the microRNA precursors from SBI s collection The HIV 1 derived pMIRNA1 pCDH vectors contain the following common features WPRE element enhances stability and translation of the CMV driven transcripts SV40 polyadenylation signal enables effici
16. ggesting that the perturbation in let 7 expression led to overexpression of RAS and thus oncogenesis Viruses which cause human disease also utilize miRNAs to regulate both host and viral gene expression Pfeffer et al Pfeffer 2004 found that human cell lines latently infected with Epstein Barr herpesvirus expressed miRNA of viral origin Computational analysis of the potential targets of these miRNA revealed predicted interactions with cellular genes involved in proliferation apoptosis immune response and other vitally important pathways Human immunodeficiency virus HIV is also predicted to express a small subset of miRNAs that have a broad range of predicted cellular targets Bennasser 2004 Conversely cellular miRNAs expressed in T cells are predicted to interact with the viral transcripts Hariharan 2005 C Tools for Functional Study of MicroRNA As the identification of novel microRNAs continues hundreds of isolated miRNAs and thousands of predicted conserved miRNAs John 2006 have been published Yet very few have had their functions experimentally validated There is an increasing need for robust methods to study the functions of each isolated or predicted microRNA The only commercially available product for the functional study of miRNAs is the collections of synthetic miRNA molecules based on predicted mature miRNA sequence Despite their optimized design criteria synthetic miRNAs underscore the importance of primary miRNA
17. inated outside of the immediate laboratory area are to be placed in a durable leak proof properly marked biohazard 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 Page 8 ver 2 082412 www systembio com MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 Protocol A Applications of SBI s MicroRNA Precursor Constructs The major advantage of SBI s microRNA precursor constructs compared to synthetic microRNA is that our constructs can be integrated into cells via the lentiviral expression system With the lentiviral system it is possible to generate stable cell lines that express specific microRNA Since microRNAs are believed to be involved in various cellular pathways these stable cell lines would provide a unique tool that allows you to link microRNA with biological functions To make a stable cell line you first have to package the construct into pseudoviral particles see Section B of Protocol After infecting your target cells with the pseudovirus containing supernatant you can isolate infected cells expressing a high level of copGFP by FACS sorting Cells that stably express specific microRNA precursors can be also used to monitor other gene expression in order to identify miRNA target genes Alternatively the microRNA precursor construct can be cotransfected with a reporte
18. mid lentivector constructs e QuantiMir small RNA quantitation system Cat RA420A 1 Easily profile miRNAs using a single cDNA synthesis step and real time qPCR e LentiMag Magnetotransduction Kit Cat LV800A 1 A novel simple and highly effective approach to increase the number of cells positively transduced with SBI s HIV and FIV based lentiviral vectors compared to the standard method of Polybrene aided transduction e 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 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 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 16 ver 2 082412 www systembio com MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 lll Licensing and Warranty Statement Limited Use License Use of the MicroRNA
19. n 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 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 888 266 5066 Toll Free 650 968 2200 outside US Page 17
20. nd 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 I Introduction and Background A Purpose of this Manual This manual provides details and information about microRNA Precursor Constructs in SBI pMIRNA1 lentivectors parent SBI vector is pCDH CMV MCS EF1 copGFP SBI cat CD511B 1 This manual does not include all the information on packaging the lentivector expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles This information is available in the user manual Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells which is available on the SBI website www systembio com Before using the reagents and material supplied with this system please read the entire manual B Overview 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 pre
21. om MicroRNA Precursor Constructs Cat s PMIRHxxxPA 1 C Packaging SBI s Cloned MicroRNA Precursor Constructs SBI s cloned microRNA precursor constructs can be efficiently packaged into VSV G pseudotyped viral particles using SBI s pPACKH1 Packaging Plasmid Mix Cat LV500A 1 The provided miRNA precursor expression plasmid was purified using a QIAGEN Endotoxin free plasmid kit to ensure maximum transfection efficiency For a detailed packaging protocol please refer to SBI s pPACK user manual Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells which is available on the SBI website www systembio com k pvsv e Construct pPACK Packaging Plasmid Mix Packaging Plasmid s NS Step 1 Co transfect 293TN cells with a lentiviral expression 203TN Ae x construct and pPACK prid Carto packaging plasmid mix Me Step 2 Collect pseudoviral particles Pseudoviral Mm and determine the titer Particles ee 3 oo 4 E Step 3 Infect target cells Target mme E e Cells NC dar 7 b V Step 4 Assay Cells e g by FACS ee analysis Ee Ro c d Phase Contrast GFP Fluoresence Mature miRNA Expression n E miRNA 205 ic 36 Lentiviral Pre miR 205 transduction taken after 48 hours Expression of mature miR 205 was validated and quantitated using ABI TaqMan assays 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI Use
22. r Manual 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 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 regulator
23. r construct that expresses putative microRNA target sequences By measuring the reporter gene expression in transfected or transduced cells the putative microRNA target gene can be confirmed 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual B Transfection of the microRNA Precursor Construct The microRNA precursor construct can be introduced together with a reporter construct into HeLa or HEK 293 cells using chemical transfection For example with these cells the Lipofectamine Reagent Invitrogen Cat 18324 111 with Plus Reagent Invitrogen Cat 11514 015 system works well Alternatively you can use your target cells for this analysis If you have already established a transfection method for your target cells use your established conditions If you do not have an established transfection protocol we recommend you compare efficiencies of several transfection procedures e g Invitrogen s Lipofectamine 2000 Cat 11668 027 Clontech s CLONfectin Cat 631301 It is important to optimize the selected transfection protocol and keep the parameters constant between testing samples and control samples Sample Transfection Data Phase Contrast GFP Fluorescence Poy yr Rx Ep ar PAS Y C s ey 293TN Cells transfected with 500 ng PreMiR 513a 1 cat PMIRH5131PA 1 using Lipofectamine 2000 Invitrogen Photos taken after 48h Page 10 ver 2 082412 www systembio c
24. sachusetts 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 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 Cont
25. vel 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 substrates 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 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 D Related Products e 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 e 293TN Human Kidney Producer Cell Line SBI Cat LV900A 1 For packaging of plas
26. y 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 I Hwang R Rebres T Roach W Seaman M I 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 ll 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 short hairpin RNAs Genes Dev 17 24 3011 6 Pooled Library For added convenience a pool of prepackaged viral particles containing the entire microRNA Precursor Clone Collection is available The pooled library can be used for high throughput phenotypic screens of microRNA function For general information
Download Pdf Manuals
Related Search