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1. HEPG2 293 T BM human hepatocellular liver carcinoma TM human embryonic kidney 100 fo 80 80 2 i S 60 8 60 oO 3 2 E 40 40 X 20 FIV based pSIF1 copGFP L 20 FIV based pSIF1 copGFP E HIV based pSIH1 copGFP E HIV based pSIH1 copGFP 0 0 0 12 3 4 5 6 7 8 9 10 11 12 0123 45 6 7 8 9 10 11 12 13 14 15 Viral Titer arbitrary units Viral Titer arbitrary units HeLa S3 H1299 human cervix carcinoma human non small cell lung carcinoma 10096 100 80 80 2 EL 9 60 8 60 E 8 E 40 3 40 e FiV based pSIF1 copGFP FIV based pSIF1 copGFP L 20 HIV based pSIH1 copGFP aL 20 E HIV based pSIH1 copGFP 0 0 0123 45 6 7 8 9 1011 1213 14 15 16 17 012 3 4 5 6 7 8 9 10 11 12 13 14 15 Viral Titer arbitrary units Viral Titer arbitrary units UMUC 3 BT 474 dopo human bladder carcinoma human breast ductal carcinoma 0 100 80 80 ey 2 g 60 8 60 o o g g Q 40 40 t aL 20 9 FIV based pSIF1 copGFP L 20 FIV based pSIF1 copGFP
2. Transduction with shRNA Library to induce phenotype ro Ate sp x om Treated Cells Selection for phenotype Cells with qu V K amp x Specific Tw x Tr Phenotype Amplification of shRNA effectors from selected cells Hybridization to Affymetrix GeneChip or sequencing Analyze with statistical software Sorted Cells v Selected ag out shRNAs Identified 100 Unsorted Cells mRNA Targets Discovered GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 B Optimize Transduction Efficiency with the copGFP Packaged Transduction Control Pantropic VSV G pseudotyped viral particles containing the lentiviral shRNA construct can be efficiently used to deliver and stably express shRNA sequences in a wide range of mammalian target cells but transduction efficiency can vary significantly depending on the target cells see Appendix A The packaged pSIH1 or pSIF1 H1 siLuc copGFP control vector can be used to estimate and optimize transduction conditions for any target cells with the GeneNet shRNA Library After transduction in target cells and integration into genomic DNA the H1 siLuc copGFP control vector stably expresses the fluorescent copGFP marker This way you can easily measure the percentage of transduced cells using fluorescent microscopy or flow cytometry and calculate copy number Expression of the copGFP reporter can be measured directly at about 72 hours after transduction The goa
3. NB41 NIH3T3 mouse neuroblastoma mouse embryonic fibroblast 100 100 80 80 2 2 g 60 g 60 o o L 2 Q 40 Q 40 E E 3 20 FIV based pSIF1 copGFP SS 20 FIV based pSIF1 copGFP E HIV based pSIH1 copGFP 8 HIV based pSIH1 copGFP 0 0 012 3 4 5 6 7 8 9 10 11 12 13 14 15 16 o 1 2 3 4 5 6 7 8 9 10 Viral Titer arbitrary units Viral Titer arbitrary units P388 mouse Lin ckit bone marrow mouse lymphocytic leukemia stem cells 100 100 FIV based pSIF1 copGFP 80 77 FIV based pSIF1 copGFP 80 8 HIV based pSIH1 copGFP o E HIV based pSIH1 copGFP a g 60 g 60 o o L E O 40 Q9 40 2 2 E a 20 d 20 0 0 o 1 2 3 4 5 6 7 8 9 10 5 10 15 20 2 30 35 40 45 50 55 Viral Titer arbitrary units Viral Titer arbitrary units Page 46 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 B Maps and Features of Single Promoter pSIH and SIF1 H1 Vectors Single marker shRNA Dual marker shRNA expression vectors expression vector pSIH1 H1 pSIF H1 pGreenPuro shRNA Vectors v shRNA Vector l P siRNA template insert sense loop antisense terminator Transcription sense w shRNA antisense Dicer sense UU siRNA antisense The shRNA template sequence is cloned into the shRNA expression cassette which is the same for both pSIH1 H1 and pSIF1 H1 cloning vectors shR
4. 22 326 330 Robinson B and Gudkov A V Genetic suppressor elements in the characterization and identification of tumor suppressor genes In Methods in Molecular Biology Tumor Suppressor Genes Pathways and Isolation Strategies Ed Wafik S E Humana Press Inc Totowa NJ 2002 222 411 434 Sui G Soohoo C Affar E B Gay F Forrester W C and Shi Y 2002 A DNA vector based RNAi technology to suppress gene expression in mammalian cells Proc Natl Acad Sci U S A 99 5515 5520 Viskers T A Koo S Bennett C F Crooke S T Dean N M and Baker B F Efficient reduction of target RNAs by small interfering RNA and RNase H dependent antisense agents J Biol Chem 2003 278 9 7108 7118 Zheng L Liu J Batalov S Zhou D Orth A Ding S and Schultz G An approach to genomewide screens of expressed small interfering RNAs in mammalian cells Proc Natl Acad Sci 2004 101 135 140 Wiznerowicz M and Trono D 2003 Conditional suppression of cellular genes lentivirus vector mediated drug inducible RNA interference J Virology 16 8957 8961 Lentiviral delivery vector reviews 888 266 5066 Toll Free 650 968 2200 outside US Page 39 System Biosciences SBI User Manual Curran MA Nolan GP Nonprimate lentiviral vectors Curr Top Microbiol Immunol 2002 261 75 105 Curran MA Nolan GP Recombinant feline immunodeficiency virus vectors Preparation and use Methods Mol Med 2002
5. 69 335 50 Loewen N Barraza R Whitwam T Saenz DT Kemler I Poeschla EM FIV Vectors Methods Mol Biol 2003 229 251 71 Naldini L Lentiviruses as gene transfer agents for delivery to non dividing cells Curr Opin Biotechnol 1998 Oct 9 5 457 63 Sauter SL Gasmi M FIV vector systems Somat Cell Mol Genet 2001 Nov 26 1 6 99 129 Genetic Screens with shRNA libraries Aza Blanc P Cooper C L Wagner K Batalov S Deveraux Q L and Cooke M P 2003 Identification of modulators of TRAIL induced apoptosis via RNAi based phenotypic screening Molecular Cell 12 627 637 Bailey S N Ali S M Carpenter A E Higgins C and Sabatini D 2006 Microarrays of lentiviruses for gene function screens in immortalized and primary cells Nature Methods 3 117 122 Berns K Hijmans E M Mullenders J et al 2004 A large scale RNAi screen in human cells identifies new components of the p53 pathway Nature 428 431 437 Bortone K Michiels F Vandeghinste N Tomme P and van Es P 2004 Functional screening of viral snRNA libraries in human primary cells Drug Discovery World Fall 20 27 Brummelkamp TR Fabius AW Mullenders J Madiredjo M Velds A Kerkhoven RM Bernards R Beijersbergen RL 2006 An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors Nat Chem Biol 2 4 202 206 Cullen LM Arndt GM 2005 Genome wide screening for gene function using RNAi in m
6. GAPDHmRNA 5 GGGCTGGCATTGCCCTCAACGACCACT 3 Ni DICER digestion sites target sequence Design of the shRNA expression cassette The shRNA template sequence is cloned into the shRNA expression cassette which is the same for both pSIH1 H1 and pSIF1 H1 cloning vectors siRNA template sequences are designed to be directionally inserted between the BamHI and EcoRI nucleotide overhangs i e sticky ends The nucleotides for the specific siRNA sequence are shown in capital letters The siRNA sense and antisense sequences flank the region coding for the loop structure In addition a terminator sequence for the RNA polymerase III is included after the antisense portion After transcription a stem loop stem siRNA molecule is produced This molecule is processed by the Dicer enzyme to generate a double stranded siRNA effector Page 52 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 D Location and Sequences of Amplification Primers pSIH1 H1 vectors 1 i CMV Fwd GNH Primer Promoter uw o 7 16 CCCCTCACCGCGGGACGTTATAAACGTACAGCGATACAC Fwd GNH Primer o ATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTA 66 AAGACCCTTTAGTGGTATTTGCACTTTACAGAAACCTAAACCCTTAGAAT 1 H1 Promoter rP Sense TAAGTTCTGTATGAGACCACTTGGATCCGNNNNNNNNNNNNNNNNNNNNN 116 AT TCAAGACATACTCTGGTGAACCTAGGCNNNNNNNNNNNNNNNNNNNNN HI Ba 96 RNA Pol Ill i d z Bio NFwd Bio Primer Anseshan Terminator
7. Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 D Data Analysis Problems 1 General Recommendations In the report file produced by the GeneNet software you can find the estimated background value Based on our experience data points with an intensity value two times greater than background may be considered as reliable data points 888 266 5066 Toll Free 650 968 2200 outside US Page 37 System Biosciences SBI User Manual IV References General references Abbas Terki Blanco Bose N Deglon Pralong W and Aebischer P 2002 Lentiviral mediated RNA interference Hum Gene Ther 13 2197 2201 Aza Blanc P Cooper C L Wagner K Batalov S Deveraux Q L and Cooke M P Identification of modulators of TRAIL induced apoptosis via RNAi based phenotypic screening Mol Cell 2003 12 627 637 Berns K Hijmans E M Mullenders J Brummelkamp T R Velds A Helmerlks M Kerkhoven R M Madiredjo M Nijkamp W Weigelt B Agami R Ge W Cavet G Linsley P S Beijrsbergen R L and Bernanrds R 2004 A large scale RNAi screen in human cells identifies new components of the p53 pathway Nature 428 431 437 Buchschacher G L and Wong Staal F 2000 Development of lentiviral vectors for gene therapy for human diseases Blood 95 2499 2504 Burns J C Friedmann T Driever W Burrascano M and Yee J K 1993 Vesicular stomatitis virus G glycoprotein pseudotyped retroviral
8. lentiviral constructs integrate into the cellular genome and each cell acquires and expresses one or a few unique shRNA library inserts Select cells with a specific phenotypic trait e g resistance to radiation apoptosis etc and expand surviving cells Alternatively select a target cell subpopulation displaying a desired phenotype by FACS or binding to Ab beads using phenotype specific markers cell morphology behavior etc Isolate total RNA and DNA from selected and control cells Amplify and label the shRNA inserts with biotin by RT PCR from total RNA isolated from the cells Alternatively you can amplify shRNA inserts from genomic DNA Remove non biotinylated sense strand of amplified shRNA inserts by treatment with Lambda exonuclease Hybridize the biotin labeled amplified ShRNA targets with an Affymetrix GeneChip Array In some cases you may alternatively clone and sequence amplified RNA inserts from selected phenotype specific clones This approach however is very time consuming and not suitable if there are a large numbers of different shRNA templates present in the selected cell population With the microarray approach it is possible to identify shRNA effectors with a weak phenotypical effect by analyzing changes in hybridization signals between control and selected target Q NewTarget ver 5 080511 User Manual Library Workflow re s Ee Infected iem EY Target Cells Treatment
9. 5 ml of washing buffer for each wash For maximum PCR product recovery elute PCR product from each column once with 22 ul of elution buffer followed by a second elution with 22 ul of elution buffer Combine all eluates for each sample into one test tube and concentrate by vacuum centrifugation to a 50 ul volume Take a 1 ul sample from each test tube dilute it in an appropriate volume of H2O and measure the yield of PCR products using a spectrophotometer at 260 nm The yield of single stranded shRNA products should be approximately 10 ug for all samples 888 266 5066 Toll Free 650 968 2200 outside US Page 31 System Biosciences SBI User Manual Positive Control DNA Experimental Samples 1 2 M 1 C 2 3 4 5 2e 3e 4e 5e M gt oes co Ist 2nd 1stPCR 2ndPCR Exo Lamda PCR PCR Treatment Analysis of shRNA insert products amplified by RT PCR from total RNA In this experiment an HIV based GeneNet Human 50K shRNA Library in pSIH1 H1 Puro was used to transduce H1299 cells 1 with Fwd GNH Rev GNH primers First PCR step E 2 6 2 NFwd Bio NRev GNH 3 NFwd Bio NRev GNH1 4 NFwd Bio NRev GNH2 5 NFwd Bio NRev GNH3 Second PCR step E 2 j 2e 3e 4e 5e Products 2 3 4 5 treated by lambda exonuclease step E 3 b C Negative control no cDNA synthesis The shRNA template recovery procedure en
10. basis 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 and foreign equivalents owned by the Dana Farber Cancer Institute Inc FIV Vector System This Product is for non clinical research use only Use of this Product to produce products for sale or for any diagnostic therapeutic clinical including pre clinical veterinary or high throughput drug discovery purpose the screening of more than 10 000 compounds per day is prohibited In order to obtain a license to use this product for these commercial purposes contact The Regents of the University of California This Product or the use of this Product is covered by U S Patent No 6 555 107 owned by The Regents of the University of California 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 re
11. to primary cell lines stem cells cells isolated from organisms blood cells tissue biopsies or even directly in model organisms mouse The high efficiency of transduction and physiological way of delivery achieved by the use of lentiviral shRNA libraries greatly facilitates complex genetic selection schemes and allows the identification of cellular targets linked directly to phenotypes e Genome wide high complexity shRNA libraries comprised of a redundant set of shRNAs 3 5 shRNAs per transcript to provide reliable knockdown for each known human or mouse gene e Ready to use shRNA libraries pre packaged as VSV G pseudoviral particle stocks that have passed stringent controls for the absence of replication competent virus contamination This significantly adds to the convenience and safety by eliminating the need for researchers to work with complicated packaging cell line technology e Post screening identification of siRNA sequences using microarrays The sequences of siRNA templates are selected according to corresponding probe sequences on the Affymetrix GeneChip Arrays Using the same sequences for the siRNA and microarray allows high throughput identification of siRNA effectors modulating a specific phenotype with the microarrays Page 4 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Lentiviral shRNA Expression Vectors Lentiviral expression vectors are the most effective vehicles for transduci
12. two nested primers one primer has biotin residues at the 5 end and another a 5 phospate group the amplified shRNA targets are labeled with biotin sense strands removed by lambda exonuclease and biotin labeled antisense strands are used as hybridization targets for Affymetrix GeneChip Arrays using standard protocols Page 26 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Notes n addition to amplifying and labeling RNA isolated from your samples you should also include a positive control using 10 ng of the Positive Control DNA that is included with the GeneNet M Library The Positive Control DNA included in the kit is the GeneNet shRNA Library in plasmid form This control can be used to optimize and troubleshoot your RT PCR and array hybridization Moreover the hybridization pattern generated from the Positive Control DNA reflects the abundance level of all ShRNA inserts in the packaged shRNA library and can therefore be used as a standardizing reference for all snRNA target samples rescued from your control and selected target cells Titanium Taq Poly is key to the success of the protocol Running all analytical gels while performing the protocol is important because this can help troubleshoot any discrepancies detected early on in the process rather than later After running the First Round there should be a bright band to indicate the quality of the product There should also o
13. 25mM HEPES pH 7 4 or in sterile PBS The total number of infection units ifu and concentration the titer were determined by measuring copy number of integrated lentiviral constructs in genomic DNA of transduced HT1080 cells using the UltraRapid Lentiviral Titering Kit and may vary for different lots of each library The exact ifu titer and volume for each GeneNet Library are indicated on the corresponding Product Analysis Certificate RT PCR primers are provided to amplify biotinylated hybridization targets comprising ShRNA inserts from total cellular RNA or alternatively from genomic DNA and to be used for hybridization with the corresponding Affymetrix GeneChip Array The sequence of the PCR primers depends on the library vector HIV based or FIV ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 based The specific sequences of the PCR primers along with the map of the amplified region can be found in the Appendix The Nested Reverse Primers have a phosphate at the 5 end for selective degradation of the sense strand in amplified shRNA targets with Lambda exonuclease e The GeneNet shRNA Library Kit is shipped on dry ice and should be immediately stored at 70 C upon receipt Avoid thawing and refreezing of pseudoviral particles Each freeze thaw cycle causes reduction of the titer by 20 30 Properly stored pseudoviral particles are stable for 6 months from the date received e The list of tar
14. 260 nm The expected yield of PCR products should be approximately 15 25 ug 3 Lambda Exonuclease Treatment a To remove sense non biotinylated strands we additionally treated all PCR products with exonuclease Lambda This exonuclease destroys the sense strand with the 5 phosphate group leaving the single stranded biotinylated antisense shRNA strand For each PCR sample from step 2 k add 20 ul of 10X ExoLambda Buffer 100 units 10 20 ul of Exonuclease Lambda New England BioLabs Cat M0262S and incubate at 37 C for 2 hours When the program is completed analyze a 1 ul sample from each tube and a 1 ul sample from each tube from Step 2 k alongside a 50 bp DNA size marker by running on a 3 agarose EtBr gel in 1X TAE to ensure that the double stranded PCR product has been degraded Purify PCR products using QIAGEN s QlAquick PCR Purification kit with the following modifications to the manufacturer s protocol e For each PCR reaction test tube add ten volumes of PB buffer 2 ml and sequentially apply 0 5 ml at a time to two QlAquick columns 888 266 5066 Toll Free 650 968 2200 outside US Page 59 System Biosciences SBI User Manual e Perform the wash step two times instead of one using 0 5 ml of washing buffer for each wash For maximum PCR product recovery elute PCR product from each column once with 22 ul of elution buffer followed by a second elution with 22 ul of elution buffer Combine all eluates
15. 2x10 ifu Cat LV201B 1 included with GeneNet shRNA Libraries in pSIF Vectors Packaged positive control FIV based lentivector allows you to measure transduction efficiency in target cells based on percent of GFP positive cells The lentivector also expresses an shRNA targeting Luciferase Global UltraRapid Lentiviral Titering Kit Cat LV961A 1 human and mouse compatible The Global UltraRapid Lentiviral Titer Kit is designed to rapidly determine the titers of pseudoviral particles that are generated with SBI s HIV and FIV lentiviral vectors or libraries It allows users to measure the copy numbers of integrated lentiviral constructs in genomic DNA of transduced target cells ShRNA Cloning and Expression Lentivectors many These HIV and FIV based single promoter shRNA cloning vectors allow you to clone and express shRNA constructs for positive control genes which are involved in your biological mechanism of interest and will be enriched for depleted in the phenotypical selection step For a list of currently available vectors please visit our website at www systembio com cDNA Cloning and Expression Lentivectors many These HIV and FIV based cDNA cloning vectors allow strong and ubiquitous expression of your gene of interest involved in your biological pathway of interest Choose from copGFP or puromycin selection markers For a list of currently available vectors please visit our website at www systembio com pGreenFir
16. 3 inactivating 3 LTR with deletion in U3 region prevents formation of replication competent viral particles after integration into genomic DNA H1 RNA promoter 3761 3977 RNA polymerase IIl promoter for expression of SiRNA insert 3979 4053 shRNA targeting Firefly Luciferase SV40 Poly A 4335 4466 Transcription termination and polyadenylation SV40 Ori 4475 4621 Allows for episomal replication of plasmid in allows or cells pUC Ori 4991 5664 C Allows for high copy replication in E coli AmpR 5809 6669 C moin resistant gene for selection of the plasmidi in E coli The notation C refers to the complementary strand 888 266 5066 Toll Free 650 968 2200 outside US Page 55 System Biosciences SBI User Manual 2 pSIF1 H1 siLuc copGFP Cat LV201B 1 Feature Location Function CMV S LTR 1415 Hybrid CMV promoter R US long terminal repeat required for viral packaging and transcription gags 762 1011 Packaging signal RRE 1012 1143 Rev response element binds gag and involved in packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1150 1391 region involved in nuclear translocation and integration of transduced viral genome CMV promoter 1394 1745 Human cytomegalovirus CMV constitutive promoter for transcription of copGFP Copepod green fluorescent protein similar to copGFP 1753 2511 regular EGFP but with brighter color as a reporter for the transfected transduced ce
17. 30 sec 18 cycles 68 C for 3 min 15 C hold 3 Run Gel When the program is completed analyze a 1 ul sample from each tube alongside a 50 bp DNA size marker by running on a 2 596 agarose EtBr gel in 1X TAE Compare your results to Figure 9 to confirm that your reactions were successful Note If the yield of expected PCR products is less than those in the positive control sample based on the intensity of the gel bands perform an additional 2 3 cycles of PCR at 94 C for 30 sec 68 C for 1 min Alternatively you can repeat the second round PCR starting from a 5 pl aliquot from step 2 e 4 PCR Purification Purify PCR products with QIAGEN s QlAquick PCR Purification kit see Section I E with the following modifications to the manufacturer s protocol e For each of the 6 PCR reaction tubes add six volumes of PB buffer and bind to a single QlAquick column e Perform the wash step twice using 0 5 ml of washing buffer for each wash Page 30 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 For maximum PCR product recovery elute PCR product from the column once with 22 ul of elution buffer followed by a second elution with 22 ul of elution buffer The total volume should be approximately 40 ul after elution e Combine all eluates from each sample into one tube The total volume should be about 260 ul Take a 1 ul sample from each test tube dilute it in an appropriate volume of TE and measure t
18. 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 Fluorescent Protein 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 Page 62 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 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 Wa
19. A Library pseudoviral particles in a water bath at 37 C Transfer the thawed particles to a laminar flow hood and keep on ice if not used immediately Dilute an appropriate amount of GeneNet shRNA library usually about 1x10 ifu with 15 ml of complete medium in order to have a final concentration of pseudoviral particles equal to the concentration of packaged copGFP control vector necessary to get MOI between 0 5 and 1 For extremely fast growing and metabolizing cell lines such as 293T use 396 FBS in the medium Add TransDux to a final concentration of 1x Caution Only open the tube containing the pseudoviral packaged GeneNet ShRNA Library in the laminar flow hood Note Gently mix the pseudovirus with the medium by rotation or inversion Do not vortex Note The remaining pseudoviral stock may be refrozen at 70 C but it will result in a loss of about 20 of the infection particles 3 Remove the culture medium from cells Infect target cells by adding the 3 ml viral stock dilutions to each of the five plates For one plate the mock transduction control add 3 ml of D MEM medium with TransDux Incubate cells at 37 C with 596 CO overnight Day 4 4 By day 4 the culture will be confluent Split 1 3 and continue incubating for 24 hours in complete D MEM Plate about 2x10 cells in a separate 6 cm plate to determine the copy number in transduced cells Day 5 72 hours after transduction 5 At this stage you c
20. B 1 SIGXXB 1 Michiels F Es H and Tomme P One step further towards real high throughput functional genomics Trends in Biotechol 2003 21 147 152 Morgan R A Cornetta K and Anderson W F 1990 Application of the polymerase chain reaction in retroviral mediated gene transfer and the analysis of gene marked human TIL cells Hum Gene Ther 1 135 149 Paddison P J Silva J M Conlin D S Sclabach M Li M Aruleba S Balija V O Shaughnessy A Gnoj L Scolbe K Chang K Westbrook T Cleary M Sachldanandam R McCombie W R Elledge S and Hannon G J 2004 A resource for large scale RNA interference based screens in mammals Nature 428 427 431 Pfeifer A Kessler T Yang M Baranov E Kootstra N Cheresh D A Hoffman R M and Verma I M 2001 Transduction of liver cells by lentiviral vectors Analysis in living animals by fluorescence imaging Mol Ther 3 319 322 Qin X F An D S Chen I S and Baltimore D 2003 Inhibiting HIV 1 infection in human T cells by lentiviral mediated delivery of small interfering RNA against CCR5 Proc Natl Acad Sci USA 100 183 188 Quinn T P and Trevor K T 1997 Rapid quantitation of recombinant retrovirus produced by packaging cell clones Biotechniques 23 1038 1044 Reynolds A Leake D Scaringe S Marshall W Boese Q and Khvorova A RNA interference mechanistic implications and rational snRNA design Nat Biotech 2004
21. DNA DNAeasy Kit QIAGEN Cat 69504 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual For Reverse Transcription of total RNA from target cells Reverse Transcriptase Recommended M MLV Reverse Transcriptase 10 U l Epicentre Cat M6125H with 10X Reverse Transcription buffer and DTT or M MLV Reverse Transcriptase 200 U ul Invitrogen Cat 28025 013 with 5X Reverse Transcription buffer and DTT dNTP set 100 mM Amersham Cat 27 2035 01 Before using mix together the four dNTP to make a final concentration of 10 mM of each dNTP For PCR Amplification of shRNA inserts Taq DNA polymerase Recommended Titanium Taq DNA Polymerase 50X Clontech Cat 639208 with 10X Titanium buffer dNTP set Amersham Cat 27 2035 01 Thermal Cycler DNA Engine MJ Research Cat PTC 200 2 5 1X TAE Agarose gel For Lambda Exonuclease treatment of biotinylated shRNA targets Lambda Exonuclease Recommended Lambda Exonuclease 10 U l New England BioLabs Cat M0262S with 10X ExoLambda buffer For Purification of amplified shRNA inserts PCR purification kit Recommended QlAquick PCR Purification Kit QIAGEN Cat 28106 For Hybridization of shRNA targets with Affymetrix GeneChip For Human 50K Libraries Human Genome U13342 0 GeneChip Array Affymetrix Cat 900470 For Mouse 40K Libraries Mouse Genome 430 2 0 GeneChip Array Affymetrix Cat 900495 Reag
22. E HIV based pSIH1 copGFP E HIV based pSIH1 copGFP 0 0 0 1 2 3 4 5 6 7 8 9 10 t1 0 1 2 3 4 5 6 7 8 9 10 Viral Titer arbitrary units Viral Titer arbitrary units 888 266 5066 Toll Free 650 968 2200 outside US Page 43 System Biosciences SBI User Manual Human Cell Lines cont d MCF 7 OVCAR 3 human breast adenocarcinoma TS human ovarian adenocarcinoma 10095 a a ae F V based pSIF1 copGFP 80 80 E HIV based pSIH1 copGFP 2 2 9 60 g 60 3 v 8 409 HET 9 40 8 v 20 FIV based pSIF1 copGFP X 20 E HIV based pSIH1 copGFP 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 8 4 5 6 7 8 9 10 Viral Titer arbitrary units Viral Titer arbitrary units K562 HL60 human chronic myelogenous leukemia human acute myeloid leukemia 100 4 10095 8096 80 FIV based pSIF1 copGFP o 4 H HIV based pSIH1 copGFP g 60 g 60 E E Q 40 8 40 2 s 20 FIV based pSIF1 copGFP 20 HIV based pSIH1 copGFP 0 j 0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 8 4 5 6 7 8 9 10 11 12 Viral Titer arbitrary uni
23. Expression Systems Guide to Packaging and Transduction of Target Cells user manual located on SBI s website www systembio com HIV Based gt GeneNet Human 50K Plasmid shRNA Library in pSIH1 H1 Puro 200 ug Cat SIG06PB 1 gt GeneNet Mouse 40K Plasmid shRNA Library in pSIH1 H1 Puro 200 ug Cat SIG22PB 1 FIV Based gt GeneNet Human 50K Plasmid shRNA Library in pSIF1 H1 Puro 200 ug Cat SI206PB 1 gt GeneNet Mouse 40K Plasmid shRNA Library in pSIF1 H1 Puro 200 ug Cat SI222PB 1 e 293TN Human Kidney Producer Cell Line SBI Cat LV900A 1 For packaging of plasmid GeneNet shRNA Libraries and lentivector constructs e pPACKH1 Lentivector Packaging Kit Cat LV500A 1 For packaging of HIV based lentivector expression constructs e pPACKF1 Lentivector Packaging Kit Cat LV100A 1 For packaging of FIV based lentivector expression constructs 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Page 16 Biosciences SBI User Manual pSIH1 H1 siLuc copGFP Packaged Positive Transduction Control 22x10 ifu Cat LV601B 1 included with GeneNet shRNA Libraries in pSIH Vectors Packaged positive control HIV based lentivector allows you to measure transduction efficiency in target cells based on percent of GFP positive cells The H1 siLuc lentivector also expresses an shRNA targeting Luciferase pSIF1 H1 siLuc copGFP Packaged Positive Transduction Control gt
24. GXXB 1 B Functional Screening with shRNA Libraries Gene silencing by small interfering double stranded RNAs siRNAs is becoming a powerful tool for functional analyses of the genes associated with specific biological processes in cells Scaling up this approach to the entire genome with shRNA libraries targeting every gene is facilitating progress in the area of functional genomics and systems biology There are two main strategies for using genome wide shRNA libraries for genetic screening experiments The first strategy relies on the development of a collection of shRNA molecules for each individual target gene with subsequent functional analysis through inactivation of a single gene at a time Though this strategy provides an efficient tool to study the functions of individual genes and can be used in combination with many biological assays it is very expensive and labor intensive for genome wide screens Despite this time consuming process this strategy was successfully applied for the functional analyses of thousands of genes based on collections of non verified or partially verified shRNAs see References Genetic Screens with shRNA libraries in Section IV of this manual These large scale projects represent the first attempts to apply global loss of function genetic screens to mammalian cells Unfortunately such projects require significant resources that are only plausible for research consortiums or medium to large sized companies
25. In the second strategy a library encoding a pooled set of shRNAs designed for all target genes is prepared introduced into a population of identical cells and a functional selection is applied Cells exhibiting the desired phenotypic changes are isolated and the siRNA constructs presumably inducing the phenotypes are recovered by PCR and identified by sequence analysis or microarray hybridization The main advantage of the second strategy is the possibility of creating a very high complexity genome wide shRNA library for all genes including ESTs in the genome with application for unbiased to any specific set of pre selected genes discovery of genes involved in specific phenotypes Moreover such pre made pooled shRNA libraries would also allow comprehensive cost effective loss of function genetic screens to be performed by small research groups The main disadvantage of genetic screens using shRNA libraries is the requirement for recipient cells with desired phenotypic changes to be selected from a pool of unaffected cells for example by selection based on cell survival appearance of specific markers or induction of reporter constructs cell morphology or behavior etc Efficient delivery and stable expression of siRNA effector molecules in a wide range of recipient cells are critical factors for knockdown technology Suppression of protein levels by exogenous synthetic siRNA or siRNA expressed from plasmid vectors is transient and the levels
26. NA template sequences are designed to be directionally inserted between the BamHI and EcoRI nucleotide overhangs e sticky ends The shRNA sense and antisense sequences flank the region coding for the loop structure In addition a terminator sequence for the RNA polymerase lll is included after the antisense portion After transcription a stem loop stem shRNA molecule is produced This molecule is processed by the enzyme Dicer to generate a double stranded siRNA effector 888 266 5066 Toll Free 650 968 2200 outside US Page 47 System Biosciences SBI User Manual 1 pSIH1 H1 Puro Vector Cat SI500A 1 Feature Locaton Function RSV 5 LTR 7 414 Hybrid RSV promoter R US long terminal repeat required for viral packaging and transcription REP EE 567 919 Packaging signal 1076 1309 Rev response element binds gag and involved in packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1798 1916 region involved in deus translocation and integration of transduced viral genome CMV promoter 1922 2271 Human cytomegalovirus CMV constitutive promoter for transcription of puromycin Puro 2279 2878 Puromycin resistant marker for selection of the transfected transduced cells Woodchuck hepatitis virus posttranscriptional WPRE 2885 3425 regulatory element enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR AU3 3564 4038 inactivating 3 LTR wi
27. NNNNNNCTTCCTGTCAGANNNNNNNNNNNNNNNNNNNNNNNNNNNTTTTT 166 NNNNNNIGAAGGACAGTCTNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAAA Loop EcoRI GAATTCWWWYCCAATTCTTCGATTCTGCTTTTTGCTTCTACTGGGTCTCT 216 CTTAAGAAAAGGTTAAGAAGCTAAGACGAAAAACGAAGA TGACCCAGAGA NRev GNH1 TACT NRev GNH2 CAGT NRev GNH3 TCAA 196 NRev GNH Universal Primer 95 CTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACC 256 GACCAATCTGGTCTAGACTCGGACCCTCGAGAGACCGATTGATCCCTTGG ili MM Rev GNH Primer 244 RNA Pol Il Terminator CACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGT GTGACGAATTCGGAGTTATTTCGAACGGAACTCACGAAGTTCATCACACA cDNA Synthesis GNH Primer jj 888 266 5066 Toll Free 650 968 2200 outside US Page 53 System Biosciences SBI User Manual Location and Sequences of Amplification Primers pSIF1 H1 vectors CMV 1 Promoter AGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATA STCTACCGACACTCCCTGTCCCCTCACCGCGGGACGTTAT Fwd GNF Primer m fGERTGRCGSTATGIGTTGHGEGIRATCACCATAAACGTGAAATGTCT 48 ARACGTACAGCGATACACAAGACCCTTTAGTGGTATTTGCACTTTACAGA H1 Promoter n TTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGA 98 ARCCTAAACCCTTAGAATATTCAAGACATACTCTGGTGAACCT NN 96 zl z P eae Bio NFwd Bio Primer NNNNNNNNNNNNNNNNNNNNNNNCTTCCTGTCAGANNNNNNNNNNNNNNN 148 NNNNNNNNNNNNNNNNNNNNNNNGAAGGACAGTCI
28. NNNNNNNNNNNNNNN Loop RNA Pol IIl Antisense Terminator EcoRI NNNNNNNNNNNTTTTTGAATTCWYWYCCAATTCTAGCGTCCATAAGCATT 198 NNNNNNNNNNNAAAAACTTAAGAAAAGGTTRAAGATCGCAGGTATTCBTAA 194 NRev GNF Universal Primer CTTTCTTTCGATAGCTTCCAGTGCTTTGTGAAACTTCGAGGAGTCTCTTT 248 TTTGAAGCTCCTCAGAGAAA F I NRev gt Rev GNF Primer 227 RNA Poi 8 GNF Terminator I GTTGAGGACTTTTGAGTTCTCCCTTGAGGCTCCCACAGATACAATAAAT CAACTCCTGAAAACTCAAGAGGGARCTCCGAGGGTGTCTATGTTATTTA Bk cDNA Synthesis GNF Primer 55 Page 54 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 E Features of the copGFP Transduction Control Vectors 1 pSIH1 H1 siLuc copGFP Cat LV601B 1 Feature Location Function ae for viral packaging and transcription gag 567919 Packagingsignal m mem Ennium packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1798 1916 region involved in ded translocation and integration of transduced viral genome Human cytomegalovirus CMV constitutive promoter for transcription of copGFP Copepod green fluorescent protein similar to copGFP 2279 3037 regular EGFP but with brighter color as a reporter for the transfected transduced cells Woodchuck hepatitis virus posttranscriptional WPRE 3044 3584 regulatory element enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR AUS 3723 426
29. Plasmid Library Viral Library 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Delivery of Packaged GeneNet shRNA Library into Target Cells Pantropic VSV G pseudotyped viral particles containing the RNA copy of the GeneNet ShRNA library can be efficiently used to deliver and stably express shRNA and reporter sequences in a wide range of mammalian target cells In order to provide guidelines for the use of lentivector delivery systems we compared transduction efficiencies of packaged HIV based and FIV based vectors in 27 different cell types Comparison of Transduction Efficiencies of FIV vs HIV in different cell lines at low MOI 12 8 6 9 2 0 E FIV based pSIF1 copGFP E HIV based pSIH1 copGFP ratio to H1299 o o zm oO WN TH ow O X4 TFT Hy O rt OO DB qo TF x OO o Beto 2 Qo o 0 On L h r ty Loo D db Y z a2 vs o m wm c Fort oO Lr L I 2 d o6 EJ s z 5 s8558 0a V 5220r158 9922 x o SHSESR9ER FES Xo Ss ow w I amp O9 OF I a a gt 805235 825 5 8 I z z SE p2 E 2s A o x Qo 000 9 F s m menoa GF G oo E o 090 op 2E 2 DEGE 6888 E E human mouse rat cat qc primary stem Ej These data clearly indicate that unlike commonly used cancer cell lines like H1299 HeLa HeK295 HepG2 etc which can be effectively transduced by lentivector constructs some cell types mou
30. SBI System Biosciences GeneNet Lentiviral shRNA Libraries Cat SI2XXB 1 SI6XXB 1 User Manual Store kit at 70 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 6 080511 contained in this user manual GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Contents l Introduction and Background Overview 2 Functional Screening with shRNA Libraries 3 GeneNet shRNA Libraries 4 Additional Supporting SBI Products and Services 14 Safety Guidelines esses 16 otocol Procedure Outline and General Comments ss 19 Control 2o cheeses ut onsectetuer eal tne x Mines A OR s S 21 moo MPD ommoogmr Ill Troubleshooting A Inefficient Transduction of Control or shRNA Library n 34 B Low Yield of ShRNA Targets eese 35 C Weak Hybridization Signals esse 36 D Data Analysis Problems essen 37 IV References 38 V Appendix A Transduction Efficiencies of Different Cell Lines with Lentivectors 43 B Maps and Features Single Promoter Vectors ssi i 47 C Design of shRNA Expression Cassette 52 D Location and Sequences of Amplification Primers 53 E Features of copGFP Transduction Control Vectors 55 F Protocol for Amplification of shRNA Targets from Genomic DNA 57 G Technical Support sess 60 VI Licensing and Warranty Stateme
31. U3 3068 3457 inactivating 3 LTR with deletion in U3 region prevents formation of replication competent viral particles after integration into genomic DNA H1 RNA promoter 3098 3312 RNA polymerase IIl promoter for expression of siRNA insert SV40 Poly A 3545 3676 Transcription termination and polyadenylation SV40 Ori 3685 3831 Allows for episomal replication of plasmid in eukaryotic cells pUC Ori 4201 4874 C Allows for high copy replication in E coli AmpR 5019 5879 C Ampicillin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand Page 50 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 2 pSIF1 H1 copGFP Vector Cat SI101B 1 Feature Location Function VEGE Hybrid CMV promoter R US long terminal repeat required for viral packaging and transcription gg 752101 Packaging signal O o packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1150 1391 region involved in nuclear translocation and integration of transduced viral genome Human cytomegalovirus CMV constitutive promoter for transcription of copGFP Copepod green fluorescent protein similar to copGFP 1753 2511 regular EGFP but with brighter color as a reporter for the transfected transduced cells Woodchuck hepatitis virus posttranscriptional WPRE 2518 3106 regulatory element enhances the stability of the viral tran
32. a Analysis Software and Gene List CD will enable you to analyze the hybridization data and create a report file in a format compatible with common spreadsheets and statistical programs The file lists the intensities of signal which correspond to the abundance level for each of the specific snRNA species in the library The Excel data can be analyzed and presented in conventional formats such as scatter plots or histograms using any of the standard statistical analysis software packages e g Systat or expression data analysis software e g Spotfire GeneSpring etc For more information please see the documentation included with the software An example of a scatter plot analysis of the representation of ShRNA inserts involved in radiation resistance in HT1080 cells transduced with a GeneNet Human 1 5K shRNA Library is shown in the Appendix 888 266 5066 Toll Free 650 968 2200 outside US Page 33 System Biosciences SBI User Manual Ill Troubleshooting A Inefficient Transduction of Packaged copGFP Transduction Control or shRNA Library into Target Cells 1 Poor infection efficiency Target cells have too high or too low density Plate fewer or more cells in order to have about 50 confluency at infection stage Target cell line may be difficult to transduce Use a higher concentration less fold dilution of pseudoviral particles Optimize the transduction protocol and use as positive control cells HT 1080 cell line W
33. ables you to amplify the entire pool of snRNA inserts from the enriched cell population or to retrieve individual shRNA templates from separate colonies selected by the phenotype specific screening protocol For most experiments microarray analysis provides the most efficient way to analyze enrichment of phenotype associated shRNAs in the complex shRNA population The CD included in the kit provides the necessary software for analysis of Affymetrix raw data in order to correlate it with the sequences of the shRNAs present in GeneNet shRNA library F Hybridize Biotin labeled shRNA Targets with GeneChip Array Hybridization of GeneChip Arrays with biotinylated shRNA targets is the most effective way to identify phenotype associated shRNAs in the wide range of biological systems The compact disc included in the kit provides the necessary software for Page 32 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 analysis of hybridization data and it contains the sequences of shRNAs present in the GeneNet shRNA library Hybridize about 10 ug minimum required amount is 6 ug of biotinylated shRNA target with the specific Affymetrix GeneChip Array required for your particular ShRNA library using the manufacturers standard protocols and recommended reagents Use Affymetrix Hybridization buffer with DMSO and hybridize at 45 C overnight The software provided with the library on the GeneNet shRNA Library Dat
34. ad 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 888 266 5066 Toll Free 650 968 2200 outside US Page 61 System Biosciences SBI User Manual 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 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
35. al volume e Mix contents by vortexing and spin the tube briefly in a microcentrifuge f Aliquot 4 ul of cDNA synthesis Master Mix into each tube from Step 1 c and mix contents by gently pipetting up and down g Add 1 ul 10 units of M MLV Reverse Transcriptase into each tube mix the contents by gently pipetting up and down and place the test tubes back in the thermal cycler h Incubate the tubes at 42 C for 1 hour in a hot lid thermal cycler i Stop the reaction by heating the tubes at 72 C for 5 min and then cool to room temperature j When the program is completed take a 10 ul aliquot from each test tube and transfer to a new 0 5 ml reaction tube For the positive control aliquot 10 ul from the Positive Control DNA into a new 0 5 ml tube 2 Amplification and Biotinylation The following procedure describes the protocol for amplification of shRNA inserts from cDNA using two rounds of PCR We have optimized the PCR cycling parameters using Clontech Titanium Taq DNA polymerase see Section I E and a hot lid thermal cycler DNA Engine MJ Research Cat 4 PTC 200 These parameters may vary with different polymerase mixes and thermal cyclers We recommend that you also perform amplification using the Positive Control DNA 10 ng that is included in the kit This control can be used to optimize and troubleshoot your RT PCR and GeneChip hybridization steps Note You will be using 10 ul of cDNA reaction from the previo
36. ammalian cells Immunol Cell Biol 83 217 23 Downward J 2004 Use of RNA interference libraries to investigate oncogenic signaling in mammalian cells Oncogene 23 8376 8383 Eggert U S Kiger A A Richter C Perlman Z E Perrimon N Mitchison T J and Field C M 2004 Parallel chemical genetic and genome wide RNAi screens identify cytokinesis inhibitors and targets PLOS Biology 2 1 8 Friedman A and Perrimon N 2004 Genome wide high throughput screens in functional genomics Cur Opin Genet Develop 14 470 476 Page 40 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Huesken D Lange J Mickanin C et al 2005 Design of a genome wide shRNA library using an artificial neural network Nature Biotechnol 23 995 1001 Leung RK Whittaker PA 2005 RNA interference from gene silencing to gene specific therapeutics Pharmacol Ther 107 222 39 Liang Z 2005 High throughput screening using genome wide shRNA libraries IDrugs 11 924 926 Moffat J Sabatini DM 2006 Building mammalian signalling pathways with RNAi screens Nat Rev Mol Cell Biol 7 177 187 Moffat J Grueneberg DA Yang X Kim SY Kloepfer AM Hinkle G Piqani B Eisenhaure TM Luo B Grenier JK Carpenter AE Foo SY Stewart SA Stockwell BR Hacohen N Hahn WC Lander ES Sabatini DM Root DE 2006 A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high content screen Cel
37. an confirm that you get a copy number close to 0 5 1 by measuring the percentage of GFP positive cells using FACS or fluorescence microscopy for shRNA libraries in the copGFP vector If you have used a GeneNet shRNA Library in the Puro vector the copy number in transduced cells 888 266 5066 Toll Free 650 968 2200 outside US Page 23 System Biosciences SBI User Manual 6 cm plate from step 4 can be easily determined using SBI s UltraRapid Lentiviral Titering Kit LV961A 1 see Section I E Alternatively the percent of stably transduced cells can be calculated based on number of puromycin resistant colonies D Screen Your Target Cells Pools of cells that are stably transduced with GeneNet shRNA library constructs can be optionally enriched before selection step by FACS copGFP vectors or by resistance to the antibiotic puromycin Puro vectors shRNA constructs are usually stably integrated into genomic DNA two days following infection Thus you can often apply an appropriate functional screening protocol 2 3 days after transduction Specific screening protocols will vary depending on the biological mechanism you are studying For general information and examples of successful genetic screening experiments we recommend that you refer to the Genetic Screens with shRNA Libraries section of the bibliography in the References section An example target screen is also shown below To review successful screens and the resul
38. ay 2 2 Cells should be between 50 to 70 confluent Aspirate medium from cells 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual 3 Combine culture medium with TransDux LV850A 1 to a 1X final concentration 4 Add virus to each well and swirl to mix Add increasing amounts of virus to different wells at varying MOls 1 2 5 10 and 20 etc to optimize the transduction Day 5 5 72 hours post transduction the viral genome will be integrated into the host cell genome Look at the cells for reporter expression if the viral construct has a reporter like GFP 6 Aspirate off medium Wash each well with PBS 7 Count the fraction of fluorescent cells by FACS analysis You may also visualize the cells for copGFP fluorescence but the results may be less accurate due to inconsistencies in counting methods Use an average of the fraction of green glowing cells in 5 10 random fields of view to estimate the overall fraction of fluorescent cells on the plate i e the fraction of infected cells Based on the dilution factor calculate the final concentration of pseudoviral particles which gives a copy number 1 C Transducing shRNA Library into Target Cells In order to maintain representation of the entire shRNA library the number of stably transduced cells used for transduction needs to be at least 10 fold greater than the complexity of the shRNA library For example you would need to
39. ble 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 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual 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 to always follow standard microbiological practices which include e Wear gloves and a lab coat when handling the lentiviral vectors pseudoviral particles or transduced cells e Always work with pseudoviral particles in a Class II laminar flow hood e Perform all procedures carefully to minimize splashes spills or the production of aerosols e Decontaminate work surfaces at least once a day or after any spill of viable material e Decontaminate all cultures stocks and other regulated wastes before disposal by an approved decontamination method such as autoclaving Materials to be decontaminated outside of the immediate laboratory area should be placed in a durable leak proofed properly marked biohazard infectious waste container and sealed for transportation from the laboratory Page 18 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 ll Protocol A P
40. cles 94 C for 30 sec 68 C for 30 sec 15 cycles 68 C for 3 min 15 C hold If you do see a specific PCR product with the expected size but its intensity is less than expected or is significantly weaker than in the positive control DNA try adding an additional 3 5 PCR cycles at 94 C 30 sec 68 C 30 sec We do not recommend doing more than 25 cycles for the first or second round PCR Cycling over 25 rounds often produces a high percentage of side products that can produce poor quality hybridization results Loss of the shRNA target during purification Repeat purification using another column or another lot of binding buffer Scale up the PCR reaction and use additional QlAquick purification columns per sample if necessary The binding capacity of one QlAquick column is 5 10 ug of PCR product If your yield is more than 5 ug of PCR product in one PCR reaction using two columns per reaction could recover more PCR product C Weak Hybridization Signals 1 Not enough biotinylated shRNA target Check the concentration and repeat the hybridization with a higher amount of biotin labeled shRNA targets shRNA target is not biotinylated Repeat PCR with another lot of NFwd Bio Primer contact SBI Poor hybridization The conditions for hybridization are not optimal The hybridization should follow standard Affymetrix procedures Follow the troubleshooting guidelines recommended by Affymetrix Page 36 ver 5 080511 GeneNet
41. e Transcriptional Reporter Lentivectors many HIV and FIV based transcriptional reporter vectors available in plasmid form or pre packaged in pseudoviral particles These vectors allow the creation of stable reporter cell lines which measure activation of specific signaling pathways and can be used as a read out system in genetic screen experiments with GeneNetTM shRNA libraries For a list of currently available vectors please visit our website at www systembio com ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 G Safety Guidelines SBI s lentiviral vectors are efficient gene transfer vehicles as used for research applications because of their stable integration in non dividing and dividing cells and long term transgene expression Along with our understanding that lentiviral vectors offer solutions for research applications biosafety concerns have uncovered risks due to insertional mutagenesis the generation of replication competent lentiviruses and vector mobilization Both HIV based and FIV 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 FIV based vectors upstream of 5 LTR in the lentivector allow eff
42. ents for standard hybridization washing and staining of Affymetrix GeneChip Arrays F Additional Supporting SBI Products and Services Custom Hybridization and Analysis for GeneNet shRNA Libraries Cat CS902A 1 You provide cell samples transduced with SBI s GeneNet shRNA Library We purify RNA DNA determine MOI generate hybridization targets hybridize them with the corresponding GeneChip microarray and provide you results of data analysis Custom shRNA Libraries Cat CS901A 1 You provide a list of the 100 50 000 genes for any organism with GenBank accession numbers We design shRNAs clone them in any of SBl s shRNA Page 14 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Lentivectors and provide you the shRNA library in plasmid and or packaged form with all necessary supporting information e Custom shRNA Constructs in Lentivectors Cat CS900A 1 You provide names of the genes with GenBank accession numbers We design shRNAs clone them in any of SBI s shRNA Lentivectors and provide you the shRNA construct in plasmid and or packaged form with all necessary supporting information e Plasmid GeneNet shRNA Libraries For production of packaged HIV or FIV based GeneNet shRNA Libraries in your cell culture facility The amount of plasmid is enough in order to produce at least 10 ifu of packaged pseudoviral particles A complete protocol is available in the Lentivector
43. eric inserts In addition all inserts have the expected sequence with less than a 2 mutation rate In addition in order to test the representation of shRNA inserts in the pseudoviral packaged shRNA library we reverse transcribed the viral RNA and amplified the shRNA inserts using flanking vector primers As a control we amplify the shRNA inserts from the plasmid library used in the packaging step Both samples were then hybridized to microarrays and compared in order to ensure representation was maintained after packaging An example of this type of analysis is in the graph on the left Furthermore we verified that each GeneNet shRNA Library can be efficiently transduced and expressed in target cells without significant loss of representation by amplifying shRNA inserts from pseudoviral RNA isolated from a packaged GeneNet shRNA library and from total RNA of target cells HT1080 transduced with the same library As seen in the sample data the packaging and transduction steps do not significantly affect representation of shRNA templates Moreover since the amplification is done using RT PCR this confirms that shRNA inserts are effectively expressed from the genomic DNA of target cells HT1080 transduced with the packaged shRNA library siRNA pool Viral Library expressed in cells opp tpn 10000 10000 1000 1000F 100 100 mmi n La pol xnl c rum iiiad ia ic iaauL 10000 100 1000 10000 100 1000
44. esis GNH Primer 10 uM 50 ul 50 ul 200 ul 200 ul 20 ul 50 ul Fwd GNH Forward PCR Primer 10 uM Rev GNH Reverse PCR Primer 10 uM NFwd Bio Nested Forward Biotinylated PCR Primer 10 uM NRev GNH Nested Reverse Universal PCR Primer 10 uM Positive Control DNA plasmid shRNA library used for packaging step 1ng ul pSIH1 H1 siLuc copGFP Packaged Positive Transduction Control gt 1x10 ifu CD with gene shRNA list and data analysis program compatible with Affymetrix data file GeneNet shRNA Libraries FIV based Cat s SI202B 1 SI206B 1 SI222B 1 500 2000 ul GeneNet shRNA Library pre packaged in pseudoviral particles 50 ul cDNA Synthesis GNF Primer 10 uM 50 ul Fwd GNF Forward PCR Primer 10 uM 50 ul Rev GNF Reverse PCR Primer 10 uM 200 ul NFwd Bio Nested Forward Biotinylated PCR Primer 10 uM 200 ul NRev GNF Nested Reverse Universal PCR Primer 10 uM 20 yl Positive Control DNA plasmid shRNA library used for packaging step 1ng ul 50 ul pSIF1 H1 siLuc copGFP Packaged Positive Transduction Control gt 1 x10 ifu 1 CD with gene shRNA list and data analysis program compatible with Affymetrix data file Page 12 Additional comments on product components GeneNet shRNA Library and pSIH1 pSIF1 H1 siLuc copGFP Packaged Positive Transduction Control are provided as frozen VSV G pseudotyped viral particles in DMEM
45. for each sample into one test tube and concentrate by vacuum centrifugation to a 50 ul volume d Take a 1 ul sample from each test tube dilute it in an appropriate volume of H2O and measure the yield of PCR products using a spectrophotometer at 260 nm The yield of single stranded shRNA products should be approximately 10 ug for all samples Please refer to Section II G for information on hybridization of biotin labeled targets with the GeneChip Array G Technical Support For more information about SBI products to download manuals in PDF format and to get vector map and sequence information visit our web site http Awww systembio com For additional information or technical assistance please call or e mail us at System Biosciences SBI 265 North Whisman Road Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail tech systembio com Page 60 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 VI Licensing and Warranty Statement Limited Use License Use of the GeneNet shRNA Library 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 SBI reserves the right to decide refund eligibility on a case by case
46. g 3 LTR with deletion in U3 region prevents formation of replication competent viral particles after integration into genomic DNA siRNA insert SV40 Poly A 4269 4400 Transcription termination and polyadenylation SV40 Ori 4409 4555 Allows for episomal replication of plasmid in lao i cells pUC Ori 4925 5598 C Allows for high copy replication in E coli AmpR 5743 6603 C moin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand 888 266 5066 Toll Free 650 968 2200 outside US Page 49 System Biosciences SBI User Manual 3 pSIF1 H1 Puro Vector Cat SI100C 1 Feature Location Function CMV 5 LTR 1 415 Hybrid CMV promoter R U5 long terminal repeat required for viral packaging and transcription gg o 762 1011 Packaging signal RRE 1012 1143 Rev response element binds gag and involved in packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1150 1391 region involved in nuclear translocation and integration of transduced viral genome CMV promoter 1394 1745 Human cytomegalovirus CMV constitutive promoter for transcription of copGFP Puro 1753 2352 Puromycin resistant marker for selection of the transfected transduced cells Woodchuck hepatitis virus posttranscriptional WPRE 2359 2947 regulatory element enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR A
47. get genes and shRNA inserts differs for each shRNA Library product This information is supplied on the compact disc included with each library kit e The Positive Control DNA included in the kit is the plasmid form of the GeneNet shRNA Library This DNA was used for production of the packaged GeneNet shRNA libraries The positive control DNA can therefore be used to optimize and troubleshoot your RT PCR and microarray hybridization steps The hybridization pattern generated from this Positive Control DNA reflects the abundance level of all shRNA inserts in the packaged library and can be used as a universal reference to compare with recovered shRNA templates from your transduced target cells E Additional Required Materials For Transduction of shRNA library into target cells e Dulbecco s Modified Eagle s Medium D MEM high glucose with sodium pyruvate and glutamine Invitrogen Cat 11995073 Fetal Bovine Serum Invitrogen Cat 16000036 Puromycin Sigma Cat P8833 Penicillin Streptomycin Invitrogen Cat 15070063 Trypsin EDTA Sigma Cat T3924 TransDux SBI Cat LV850A 1 Tissue Culture Plates and Related Tissue Culture Supplies For Purification of total RNA and genomic DNA from target cells e For simultaneous purification of total RNA and genomic DNA TRizol Reagent Invitrogen Cat 15596 026 e For purification of total RNA RNeasy Mini Kit QIAGEN Cat 74104 e For purification of Genomic
48. he yield of PCR products using a spectrophotometer at 260 nm The expected yield of PCR products should be approximately 15 25 ug 5 Lambda Exonuclease Treatment Notes Overdigestion with lambda exonuclease will lead to product degradation We also recommend the user to optimize the time on this step To remove the sense non biotinylated strand we additionally treat all PCR products with exonuclease Lambda This exonuclease destroys the sense strand with the 5 phosphate group leaving the single stranded biotinylated antisense shRNA strand a b d For each PCR sample from step 2 k into a 1 5 ml tube add 260 ul of the purified PCR product 34 ul of 10x ExoLambda buffer 39 5 pl of Lambda Exonulease 197 6 Units New England BioLabs Cat M0262S and incubate at 37 C for 2 hours When the program is completed analyze a 1 ul sample from each tube and a 1 ul sample from each tube from Step 2 k alongside a 50 bp DNA size marker by running on a 3 agarose EtBr gel in 1X TAE to ensure that the double stranded PCR product has been degraded Figure 9 shows results of this analysis Purify PCR products using QIAGEN s QlAquick PCR Purification kit with the following modifications to the manufacturer s protocol e For the lambda exonuclease reaction 330 ul add ten volumes of PB buffer 3 4 ml and sequentially apply 0 5 ml at a time to three QlAquick columns e Perform the wash step two times instead of one using 0
49. icient Tat independent production of viral RNA reducing the number of viral genes that are used in this system e Number of lentiviral genes necessary for packaging replication and transduction is reduced to three gag pol rev e The corresponding proteins are expressed from different plasmids that lack packaging signals The packaging plasmids share no significant homology to any of the expression lentivectors pVSV G expression vector or any other vector in order 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 Produced pseudoviral particles will carry only a copy of your expression construct The choice of SBI s lentiviral system for experimental studies is driven by functional considerations including increased productivity and transduction efficiency The design of SBI s biosafe vectors has benefited researchers allowing them to conduct experimental studies with lower risk Currently SBl s vectors combine improved safety features that decrease the risk of recombination and vector mobilization with increased transduction efficiency Despite the above safety features use of HIV based vectors falls within NIH Biosafety Level 2 criteria due to the potential biohazard risk of possi
50. l 124 6 1283 98 Ngo VN Davis RE Lamy L Yu X Zhao H Lenz G Lam LT Dave S Yang L Powell J Staudt LM 2006 A loss of function RNA interference screen for molecular targets in cancer Nature Mar 29 Paddison J P Silva J M Conklin D S et al 2004 A resource for large scale RNA interference based screens in mammals Nature 428 427 431 Paddison PJ Schlabach MR Sheth N Bradshaw J Burchard J Kulkarni A Cavet G Sachidanandam R McCombie WR Cleary MA Elledge SJ Hannon GJ 2005 Second generation shRNA libraries covering the mouse and human genomes Nat Genet 37 1281 8 Poulin G Nandakumar R Ahringer J 2004 Genome wide RNAi screens in Caenorhabditis elegans impact on cancer research Oncogene 23 8340 8345 Sachse C Echeverri CJ 2004 Oncology studies using shRNA libraries the dawn of RNAi based genomics Oncogene 23 8384 8391 Sachse C Krausz E Kronke A et al 2005 High throughput RNA interference strategies for target discovery and validation by using synthetic short interfering RNAs Functional genomic investigations of biological pathways Methods in Enzymology 392 242 277 Silva J Chang K Hannon G J and Rivas F V 2004 RNA interference based functional genomics in mammalian cells reverse genetics coming of age Oncogene 23 8401 8409 Silva JM Li MZ Chang K Ge W Golding MC Rickles RJ Siolas D Hu G Paddison PJ Schlabach MR Sheth N Bradshaw J Burchard J Kulka
51. l of transduction optimization experiments is to find the concentration of pseudoviral particles which yields a copy number between 0 5 and 1 Within this range you would expect that each cell that has been successfully transduced contains only one copy of a given shRNA construct Above this range copy number gt 1 successfully transduced cells may express more than one introduced shRNA construct To determine the concentration of pseudoviral particles required to provide a copy number between 0 5 and 1 for your particular target cells you should do several transductions with different concentrations of packaged copGFP transduction control Based on the percentage of GFP positive cells determine the transduction efficiency Use this simple guideline to convert the percentage of GFP positive cells to copy number The range highlighted in yellow is the target range for copy number GFP positive cells 10 20 30 40 50 60 70 80 90 gt 90 Copy number 0 1 0 23 0 36 0 51 0 7 0 93 1 22 1 64 2 3 52 5 Please note that copy number cannot be reliably calculated if the percentage of transduced cells is more than 90 Caution You are working with infectious pseudovirus at this stage Please follow the recommended guidelines for working with BSL 2 class viruses see Section I G for more details Day 1 1 Plate 50 000 cells per well in a 24 well plate in cell culture medium D
52. lls Woodchuck hepatitis virus posttranscriptional WPRE 2518 3106 regulatory element enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR AU3 3227 3682 inactivating 3 LTR with deletion in U3 region l prevents formation of replication competent viral particles after integration into genomic DNA H1 RNA promoter 3257 3471 RNA polymerase IIl promoter for expression of SiRNA insert 3481 3542 shRNA targeting Firefly Luciferase SV40 Poly A 3770 3901 Transcription termination and polyadenylation SV40 Ori 3910 4056 Allows for episomal replication of plasmid in eukaryotic cells pUC Ori 4426 5099 Allows for high copy replication in E coli C AmpR 5244 6104 C Ampicillin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand Page 56 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 F Protocol for Amplification of shRNA Targets from Genomic DNA Alternative to Section II E The following protocol describes the amplification of shRNA inserts from genomic DNA of target cells transduced with the GeneNet shRNA library We have optimized the PCR cycling parameters using Clontech Titanium Taq DNA polymerase see Section I F and a hot lid thermal cycler DNA Engine MJ Research Cat PTC 200 These parameters may vary with different polymerase mixes and thermal cyclers We recommend tha
53. m Step 2 a and place them in the hot lid thermal cycler e Commence thermal cycling using the following program 94 C for 2 min 94 C for 30 sec 68 C for 1 min 20 cycles 68 C for 3 min 15 C hold f When the program is completed analyze a 5 ul sample from each tube alongside a 50 bp DNA size marker by running on a 2 596 agarose EtBr gel in 1X TAE Compare your results to Figure 13 to confirm that your reactions were successful Aliquot 1 ul from each tube into four new 0 5 ml reaction tubes You will need about 4 PCR reactions per sample to obtain enough biotin labeled ShRNA target about 10 ug for hybridization with a GeneChip Array g Prepare a Second Round PCR Master Mix for all reaction tubes plus one additional tube using the following proportions Combine the components in the order shown Per Tube 66 ul Deionized H2O 10 ul 10X Titanium Taq PCR buffer 2 ul 50X dNTP mix 10 mM of each dNTP 10 ul NRev GNF GNH Nested Reverse Universal Primer 10 uM 10 ul NFwd Bio Nested Forward Biotinylated PCR Primer 10 uM 2 ul 50X Titanium Taq DNA polymerase 100 ul Total volume h Mix contents by vortexing and spin the tubes briefly in a microcentrifuge i Aliquot 100 ul of the PCR Master Mix into each tube with the 1 ul aliquot from Step 2 f and place them in the hot lid thermal cycler j Commence thermal cycling using the following program 94 C for 2 min 50 C for 2 min 68 C for 1 min 1 cycle 94 C f
54. mplification Non optimal PCR conditions After the first round of PCR you may see a weak specific band or weak smear depending on the target RNA sample However the second amplification should produce a clear band with minimal smearing If this defined band is not present you may need to optimize the PCR The yield and quality of PCR products depends significantly on the quality of PCR reagents amplification parameters PCR machine and quality or nature of your cDNA samples Always run PCR of your target samples alongside with the Positive Control DNA plasmid shRNA library and negative control cDNAs It is very critical to use hot start Taq DNA polymerase with high enzymatic activity and previously test other PCR reagents using positive controls included in the manufacturer s kit 888 266 5066 Toll Free 650 968 2200 outside US Page 35 System Biosciences SBI User Manual If after optimizing the PCR reaction you continue to generate a smear after the second round or in the negative control RNA try using a touchdown PCR protocol in the first round of PCR by starting the cycling with a higher annealing temperature than specified in the standard protocols then gradually reducing the annealing temperature in successive cycles until the recommended temperature is reached For example try the following parameters 94 C for 2 min 94 C for 30 sec 72 C for 30 sec 5 cycles 94 C for 30 sec 70 C for 30 sec 5 cy
55. ng and stably expressing different effector molecules shRNA cDNA DNA fragments antisense ribozymes etc in almost any mammalian cell including non dividing cells and whole model organisms Cann 2000 As with standard plasmid vectors it is possible to introduce lentiviral effector constructs in plasmid form into the cells with low to medium efficiency using conventional transfection protocols However by packaging the lentiviral snRNA vector construct into viral particles you can obtain highly efficient transduction and heritable expression of shRNA even with the most difficult to transfect cells such as primary stem and differentiated cells Endogenously expressed shRNA effectors provide long term silencing of the target gene and allow the researcher to generate cell lines and transgenic organisms with a stable knockdown phenotype for functional studies Moreover lentiviral delivery does not produce the non specific cell responses typically associated with chemical transfection or use of an adenoviral delivery system Gould 2003 Cann 2000 SBI offers GeneNet shRNA libraries constructed in both HIV based and FIV based lentivectors SBs lentivectors are a third generation of lentivectors developed for gene therapy applications Poeschla 2003 Sodroski 1997 1999 Federico 2003 Heiser 2004 Machida 2003 The lentiviral expression vector contains the genetic elements LTR GAG RRE cPPT WPRE required for packaging transducti
56. nly be one band after the First Round Overcycling will lead to dimers forming A negative control can also be included with your samples The negative control should contain RNA isolated from target cells that have not been transduced with the GeneNet library 1 Reverse Transcription cDNA Note The following protocol is optimized for the enzymes and reagents recommended in Section I E specifically Epicentre s M MLV Reverse Transcriptase and 10X reaction buffer Other enzymes may require somewhat different conditions a Foreach sample combine the following reagents in a 0 5 ml reaction tube 5 15 ul Total RNA sample 5 10 ug use up to 50 ug if possible for better results 1 ul CDNA Synthesis GNF GNH Primer 10 uM Deionized H2O add up to 16 ul final volume 16 ul Total volume 50 100 ul if possible for better results Use 5 ug if the RNA concentration is low b Mix contents and spin the tubes briefly in a microcentrifuge 888 266 5066 Toll Free 650 968 2200 outside US Page 27 System Biosciences SBI User Manual c Incubate the tubes at 72 C in a hot lid thermal cycler for 2 min and then reduce the temperature to 42 C d Prepare a cDNA synthesis Master Mix for all reaction tubes plus one additional tube using the following proportions Combine the components in the order shown Per Tube 2 ul 10X Reverse Transcriptase Buffer 1 ul DTT 100 mM 1 ul dNTP mix 10 mM of each dNTP 4 ul Tot
57. nt f CCfOO LL 61 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Introduction and Background A Overview This manual provides information describing genetic screening with System Biosciences s SBl s GeneNet shRNA libraries cloned in Lentiviral Expression Vectors and pre packaged in VSV G pseudotyped viral particles Specifically it provides recommendations and instructions on how to transduce packaged GeneNet shRNA libraries into target cells select target cells with a specific phenotype and identify shRNAs and corresponding target genes which induce the specific phenotype Before using the reagents and material supplied with this product please read the entire user manual Please refer to the associated Product Analysis Certificate PAC for Viral titers of the Libraries and for the Positive transduction control ShRNAs are short hairpin RNAs that have a sequence of RNA that makes a tight hairpin turn The shRNA hairpin structure is cleaved by Dicer into siRNA which is then binds to the RNA induced silencing complex RISC This complex binds to and cleaves mRNAs to which the corresponding siRNA hybridizes ShRNAs and the resulting siRNAs can be used to silence gene expression via RNA interference Lentivirus Infection DICER Processing Target mRNA cleaved and knocked down Page 2 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SI
58. of targeted gene products typically recover in several days following transfection Lockhart 1996 Lorens 2001 Michiels 2003 In order to achieve long term permanent levels of siRNA in the cell stable transcription of shRNA can be achieved by viral shRNA constructs integrated into genomic DNA of target cells From a 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual practical standpoint lentiviral vectors are an optimal delivery system for stable and effective up to 10096 transduction of gene specific RNA interference constructs and complex shRNA libraries into recipient cells see Appendix Lentiviral Delivery Vectors Based on lentiviral delivery technology SBI has developed a novel research tool for genetic screen experiments the genome wide lentiviral snRNA library C GeneNet shRNA Libraries The next generation of user friendly genetic screening technology that includes genome wide shRNA libraries has been developed at SBI with several novel features that significantly extend the application of this technology for high throughput functional genomics studies e Biosafe third generation lentiviral HIV and FIV based shRNA Vectors with puromycin selection or copGFP reporter and the RNA polymerase III H1 promoter shRNA expression cassette for the expression of shRNA constructs e Lentiviral shRNA transduction system that significantly extends the application of genetic screens
59. on stable integration of the expression constructs into genomic DNA It also contains the siRNA effector sequences driven by the H1 promoter Puromycin resistance GFP expression or both is driven by the CMV promoter The shRNA constructs packaged in pseudoviral particles can transduce target cells and express siRNA and reporter molecules but they cannot replicate within target cells because the viral structural genes are absent and the LTRs are designed to be self inactivating upon transduction Single marker shRNA Dual marker shRNA expression vectors expression vector RSV 5 LTR pSIH1 H1 pSIF H1 pGreenPuro shRNA Vectors shRNA Vector pUC ORI EcoRI BamHI T2A peptide Y SV40 ORI SV40 poly A 3 ALTR H1 wPRE 3 ALTR H1 WPRE Puro 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 1 P siRNA template insert sense loop antisense terminator Transcription sense shRNA antisense Dicer sense UU sj UU siRNA antisense Design of shRNA templates Despite the development of many algorithms for prediction of functional synthetic shRNAs Vickers 2003 Khvorova 2003 Reynolds 2004 the selection of efficient snRNA sequences that target mRNA still remains a challenging problem There is no reliable algorithm to predict the efficacy of different siRNAs The principal prediction criteria which are used to select siRNA sequences that most likel
60. or 30 sec 68 C for 30 sec 18 cycles 68 C for 3 min 15 C hold Page 58 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 k When the program is completed analyze a 1 ul sample from each tube alongside a 50 bp DNA size marker by running on a 2 596 agarose EtBr gel in 1X TAE Compare your results to Figure 13 to confirm that your reactions were successful If the yield of expected PCR products is less than those in the positive control sample based on the intensity of the gel bands perform an additional 2 3 cycles of PCR at 94 C for 30 sec 68 C for 1 min Alternatively you can repeat the second round PCR starting from a 5 pl aliquot from step 2 f Purify PCR products with QIAGEN s QlAquick PCR Purification kit see Section I E with the following modifications to the manufacturer s protocol For each PCR reaction test tube add six volumes of PB buffer and bind to a single QlAquick column e Perform the wash step two times instead of one using 0 5 ml of washing buffer for each wash For maximum PCR product recovery elute PCR product from the column once with 22 ul of elution buffer followed by a second elution with 22 ul of elution buffer Combine all four eluates from each sample into one test tube The total volume will be about 160 ul Take a 1 ul sample from each test tube dilute it in an appropriate volume of TE and measure the yield of PCR products using a spectrophotometer at
61. propriate restriction enzymes and ligated to the corresponding linearized cloning vector 4 The ligated shRNA library was then transfected into competent E coli cells grown as independent colonies on LB agar plates and the total shRNA library in plasmid DNA form was purified from the pool of independent ampicillin resistant bacterial colonies 5 The pseudoviral packaged shRNA library was then produced by co transfection of the plasmid shRNA library with the pPACK Packaging Plasmid mix into 293TN cells 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Gene Specific Segment Target pande uiis UN mRNA shRNAs 5 at mmi m AAAAAAA shRNA Library R Construction GeneChip Design amp Synthesis Oligonucleotide synthesis of shRNA sequences on chip Custom Synthesis Oligo MicroArray Oligonucleotide detachment Oligo pool of ShRNAs Amplification gt R Oligo of shRNAs R amplification shRNA pool Packaged Lentiviral High throughput Lentiviral cloning packaging shRNA Library Cloning amp Packaging C Lentivector Lentiviral based ShRNA Plasmid Library Page 8 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 6 Quality control analysis of constructed shRNA libraries was performed by sequence analysis of randomly selected clones gt 20 from each library Sequencing results show an insert rate gt 90 with lt 10 concatem
62. rni A Cavet G Sachidanandam R McCombie WR Cleary MA Elledge SJ Hannon GJ 2005 Second generation shRNA libraries covering the mouse and human genomes Nat Genet 37 11 1281 8 Sugimoto A 2004 High throughput RNAi in Caenorhabditis elegans genome wide screens and 888 266 5066 Toll Free 650 968 2200 outside US Page 41 System Biosciences SBI User Manual functional genomics Differentiation 72 81 91 Vanhecke D and Janitz M 2005 Functional genomics using high throughput RNA interference Drug Discov Today 10 205 212 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 2006 A genetic screen implicates miRNA 372 and miRNA 373 as oncogenes in testicular germ cell tumors Cell 124 6 1169 81 Willingham AT Deveraux QL Hampton GM Aza Blanc P 2004 RNAi and HTS exploring cancer by systematic loss of function Oncogene 23 51 8392 400 Zheng L Liu J Batalov S Zhou D Orth A Ding S and Schultz P 2004 Proc Natl Acad Sci 101 135 140 Page 42 ver 5 080511 GeneNet Lentiviral shRNA Libraries V Appendix A Transduction Efficiencies of Different Cell Lines with Increasing Relative Concentration of Viral Particles for HIV based and FIV based Lentivectors Cat s SI2XXB 1 SIGXXB 1 Human Cell Lines
63. robe sequences share considerable similarity with efficient siRNA sequences Moreover using sequences similar to the probes on the GeneChip Array enables the use of the microarray as a simple readout tool for analysis of siRNA recovered from selected cell populations Construction and Quality Control of shRNA libraries 1 We selected a set of target genes for each GeneNet Library e g for the Human 50K library we selected all human genes including ESTs about 47 000 transcripts represented on the GeneChip Human Genome U133 2 Array 2 For each target gene we designed 3 5 shRNA template oligonucleotides that express 27 mer siRNAs targeting each of the mRNA sequences The shRNAs were designed based on criteria developed by SBI for selection of the most efficient siRNAs Since our algorithm yields about 50 functional siRNA sequences 3 5 shRNA per target mRNA should silence about 90 of target genes for the library Each target sequence was also designed to hybridize with probe oligos on Affymetrix GeneChip Arrays and have additional 5 and 3 flanking sequences for directional cloning into a lentiviral snRNA expression vector Gene Specific Segment M M MM siRNAs e N Selection amp siRNA GeneChip Library Construction probes 3 After synthesis the shRNA template oligonucleotides were amplified by PCR using primers complementary to the additional flanking 5 and 3 sequences digested with the ap
64. rocedure Outline and General Comments GeneNet shRNA libraries provide a high throughput functional genomics approach that focuses on the identification of genes responsible for various biological processes For general information and background on working with lentiviral technology we recommend the General Reviews listed in the Reference Section particularly Cann 2000 and Buchschacher et al 2000 The diagram below outlines the general steps required for the discovery of genes modulating a specific phenotype with the pre made GeneNet shRNA library including transduction into target cells selection of cells with desired phenotype and identification of phenotype inducing shRNAs and corresponding target genes by hybridization of amplified shRNA cassettes with a GeneChip Array Some key terms used in the protocol MOI multiplicity of infection The ratio of infectious pseudoviral particles ifu to the number of cells being infected IFU cells MOI IFU ml infectious units per ml The relative concentration of infection competent pseudoviral particles Transduction Efficiency The average copy number of expression constructs per genome of target cell in the infected population 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI The overall protocol includes the following steps 1 Page 20 Transduce target cells with the GeneNet lentiviral shRNA library provided by SBI The
65. rong amount of TransDux added during infection stage TransDux is provided as a 5x solution Loss of pseudoviral titer during storage Ensure storage of the copGFP Packaged Transduction Control stock and packaged GeneNet shRNA Library at 70 C Each freeze thaw cycle causes reduction of the titer by 20 30 Use a fresh stock for transduction Do not keep the stock longer than 6 12 months Volume of infecting supernatant is too high Keep the volume as low as possible to achieve maximal adsorption of viral particles to the cells 2 Transduction affects target cell viability Packaged copGFP Control or GeneNet shRNA Library affects target cell growth Use a shorter transduction time to minimize the toxic effect to the target cells Compare toxicity of HIV based and FIV based control constructs which may be different for your target cells Try replacing with a similar target cell type Polybrene is toxic for target cells Use TransDux instead of Polybrene 3 Noexpression of copGFP reporter or ShRNAs in target cells The CMV promoter or H1 U6 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 Page 34 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 B Low Yield of shRNA Targets 1 General Recommendations The protocol for generating biotin labeled shRNA targets includes four main steps reve
66. rranty 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 2011 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 63
67. rse transcription first round PCR second round PCR and lambda exonuclease treatment It has been optimized using the specific reagents and kits specified We recommend reading both the manufacturers protocols for the respective reagents and our protocol before doing target preparation experiments For more detailed troubleshooting of each enzymatic step you should refer to the manufacturer s protocol To effectively troubleshoot the overall shRNA target preparation and hybridization and identify possible problem steps it is important to run in parallel a positive control using the Positive Control DNA included with the library kit and a negative control using RNA purified from target cells that were not transduced with the shRNA library It is critical to analyze samples from each of the enzymatic steps on an agarose gel alongside the positive and negative controls as references 2 Poor Efficiency of Reverse Transcription RNA is of low quality or impurities which inhibit reverse transcriptase If you have not already done so analyze the quality of total RNA by gel electrophoresis If you used QIAGEN RNeasy purification try purifying RNA with TRizol If you still have a problem with the RNA sample from target cells or cannot amplify PCR product from control RNA but you can amplify shRNA inserts from positive control DNA try another lot or supplier of reverse transcriptase 3 Low yield of PCR product or high level of non specific a
68. scripts Required for viral reverse transcription self 3 ALTR AU3 3227 3616 inactivating 3 LTR with deletion in U3 region prevents formation of replication competent viral particles after integration into genomic DNA H1 RNA promoter 3257 3471 RNA polymerase IIl promoter for expression of siRNA insert SV40 Poly A 3704 3835 Transcription termination and polyadenylation SV40 Ori 3844 3990 Allows for episomal replication of plasmid in eukaryotic cells pUC Ori 4360 5033 Allows for high copy replication in E coli C AmpR 5178 6038 C Ampicillin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand 888 266 5066 Toll Free 650 968 2200 outside US Page 51 System Biosciences SBI User Manual C Design of the Cloning and Expression Cassette for pSIH1 H1 and pSIF1 H1 Vectors shRNA Expression Cassette 1 Loo Terminator H1 Promoter A P Sense P Antisense M J 5 gagaccacttgGATUC GTTGGTATTGCTCTCAATGACCACTCTTCCTGTCAGAAGTGGTCGTTGAGGGCAATGCCAGCCCTTTTTGaattc 3 3 ctctggtgaaccta A CCAACCATAACGAGAGTTACTGGTGAGAAGGACAGTCTTCACCAGCAACTCCCGTTACGGTCGGGAAAAACTTAAg 5 d EcoRI BamHI Transcription atgc Vector Sequence n o ATGC shRNA Template ute Sequence GAPDH shRNA s Ged usc nuucc cucaaUGACCACU u 3 transcript 3 uuCCCGACCGUAACGGGAGUUGCUGGUGA g G U mismatches introduced g c a in sense strand to increase shRNA construct stability
69. se Lin ckit bone marrow P19 PBMC HL60 P388 are more resistant to infection More efficient transduction of resistant cell types may be possible by using a higher concentration of pseudoviral particles per cell in order to achieve the same MOI but not in all cases It is important to mention that HIV based and FIV based lentivectors have different tropism For example the FIV based shRNA constructs are more effective at infecting several of the tested mouse cell lines P19 NB41 NIH3T3 P388 and some of the blood cells MOLT 4 K562 T cells from AML patient The HIV based system is more effective at infecting stem and primary cells HUVEC bone marrow adipose Page 10 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Pseudotyped lentiviruses have been successfully used to infect many other cell types including neuronal dendritic endothelial retinal pancreatic hepatic aortic smooth muscle cells airway epithelia skin fibroblasts macrophages etc Lentivectors have also been used successfully for in vivo delivery and expression of transgenes in muscle brain airway epithelium liver pancreas retina and skin For a more complete list of cells or tissues which have been successfully transduced with lentivectors please see the Appendix Section A D Product Description and List of Components The table below outlines the general features of the available GeneNet shRNA Libraries and indica
70. t you also perform amplification using 10 ul of Positive Control DNA This control can be used to optimize and troubleshoot your PCR and GeneChip hybridization steps 2 a C Purify Genomic DNA For each fraction of selected and reference cells detach cells from plates collect and wash in PBS by centrifugation Follow standard protocols for purification of genomic DNA For most cell lines and tissue samples we recommend using TRIzol Reagent Invitrogen Cat 15596 026 Measure the concentration by measuring the absorbance at 260 nm Amplify shRNA Targets For each sample aliquot 5 ug 5 20 ul of genomic DNA from step F 1 and 10 ul of Positive Control DNA and transfer to new 0 5 ml reaction tubes In each test tube adjust the volume to 20 ul by adding the necessary volume of deionized water Prepare enough First Round PCR Master Mix for all reaction tubes plus one additional tube by combining the following components in the order shown Per Tube 62 ul Deionized H2O 10 ul 10X Titanium Taq PCR buffer 2 ul 50X dNTP mix 10 mM of each dNTP 2 ul Fwd GNF GNH Reverse PCR Primer 10 uM 2 wl RevGNF GNH Forward PCR Primer 10 uM 2 ul 50XTitanium Taq DNA polymerase 80 ul Total volume Mix contents by vortexing and spin the tube briefly in a microcentrifuge 888 266 5066 Toll Free 650 968 2200 outside US Page 57 System Biosciences SBI User Manual d Aliquot 80 ul of the PCR Master Mix into each tube fro
71. tem cells donor acute myelogenous leukemia 100 80 80 2 2 Q 60 60 o o g g 3 4096 3 40 FIV based pSIF1 copGFP FIV based pSIF1 copGFP 8 20 EH HIV based pSIH1 copGFP S 20 Hi HIV based pSIH1 copGFP 096 0 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 55 Viral Titer arbitrary units Viral Titer arbitrary units adipose tissue human mesenchymal stem cells donor 100 80 2 D 60 o E g 9 40 a 20 FIV based pSIF1 copGFP HIV based pSIH1 copGFP 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Viral Titer arbitrary units 888 266 5066 Toll Free 650 968 2200 outside US Page 45 System Biosciences SBI User Manual Mouse Cell Lines RAW 264 7 P19 mouse leukaemic monocyte macrophage mouse embryo teratocarcinoma 100 100 80 80 o 2 3 60 g 60 o Ej 3 O 40 O 40 2 2 E 3 20 FIV based pSIF1 copGFP X 20 l HIV based pSIH1 copGFP 0 REE EE EH 0 HHH o i 2 3 4 5 6 7 8 9 10 012 34 56 7 8 9 10 11 12 13 14 15 Viral Titer arbitrary units Viral Titer arbitrary units
72. teps if 10 ug for hybridization with a GeneChip Array Contents in tubes may also be combined to obtain the desired 10 ug Note If you use 6 ul in 6 tubes you should still have 89 ul remaining in case you need to go back and repeat this step later Make sure to save this until you are sure that the reactions have been successful g In the Second Round PCR prepare a Second Round PCR Master Mix for all reaction tubes plus one additional tube using the following proportions Combine the components in the order shown 888 266 5066 Toll Free 650 968 2200 outside US Page 29 System Biosciences SBI User Manual Per Tube 6 tubes per sample now Biotin Label 66 ul Deionized HO 10 ul 10X Titanium Taq PCR buffer 2 ul 50X dNTP mix 10 mM of each dNTP 10 ul NRev GNF GNH Nested Reverse Universal Primer 10 uM 10 ul NFwd Bio Nested Forward Biotinylated PCR Primer 10 uM 2 ul 50X Titanium Taq DNA polymerase Do not use any alternatives 100 ul Total volume per tube h Mix contents by vortexing and spin the tubes briefly in a microcentrifuge i Aliquot 100 ul of the PCR Master Mix into each tube with the 1 ul aliquot from Step 2 e and place them in the hot lid thermal cycler Note This will give a total volume of 101 ul of Master Mix per tube 6 tubes total j Commence thermal cycling using the following program 94 C for 2 min 50 C for 2 min 68 C for 1 min 1 cycle 94 C for 30 sec 68 C for
73. tes the compatibility of each library with the latest version of the Affymetrix GeneChip Array www affymetrix com The most updated list of shRNA libraries available in different vectors can be found on SBl s website at www systembio com catat E eco conplent ft Canech irse MRNAS SI206B 1 Human 50K P921 200000 2x 107 G U S9 38 500 47 400 SI222B 1 Mouse 40K io ae 150 000 2x10 MG 430 2 0 34 000 39 000 SI606B 1 Human 50K PSIHTH1 200000 2x 107 MODTS9 38 500 47 400 SI622B 1 Mouse 40K aua 150 000 2x107 MG 430 2 0 34 000 39 000 The shRNA libraries are provided in ready to use pre packaged in VSV G pseudotyped viral particle format or as plasmid library which you can package in pseudoviral particles in your cell culture facility Depending on the complexity of the library different amounts of pseudoviral particles infection units or ifu are provided in the kit The GeneNet shRNA Library Kits provide enough VSV G pseudotyped pre packaged shRNA library for 2 3 transductions for the most commonly used cell lines with an MOI of 1 2 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual Packaged GeneNet shRNA Library Components GeneNet shRNA Libraries HIV based Cat s SI602B 1 SIG06B 1 SI622B 1 500 2000 ul GeneNet shRNA Library pre packaged in pseudoviral particles 50 ul cDNA Synth
74. th deletion in U3 region l prevents formation of replication competent viral particles after integration into genomic DNA H1 RNA promoter 3602 3818 RNA polymerase IIl promoter for expression of SiRNA insert SV40 Poly A 4110 4241 Transcription termination and polyadenylation SV40 Ori 4250 4396 Allows for episomal replication of plasmid in ES cells pUC Ori 4766 5439 C Allows for high copy replication in E coli AmpR 5584 6444 C mpi resistant gene for selection of the plasmidi in E coli The notation C refers to the complementary strand Page 48 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 2 pSIH1 H1 copGFP Vector Cat SI501A 1 Feature Location Function on aa for viral packaging and transcription gag 567 919 Packagingsignal me one Papoea packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1798 1916 region involved in nuclear translocation and integration of transduced viral genome Human cytomegalovirus CMV constitutive promoter for transcription of copGFP Copepod green fluorescent protein similar to copGFP 2279 3037 regular EGFP but with brighter color as a reporter for the transfected transduced cells Woodchuck hepatitis virus posttranscriptional WPRE 3044 3584 regulatory element enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR AU3 3723 4197 inactivatin
75. ting publications please visit SBl s website http www systembio com rnai libraries human genome wide Zproduct 37 tab 1 3 Although the specific protocol and controls may be different depending on the cell type functional assay and selection protocol e g FACS apoptosis induction toxic chemical survival etc it is critical to carefully design your experiment in order to generate statistically significant data Page 24 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Transduction of shRNA Library O i Infected ee y Infected Target Cells Amplification amp Hybridization Analyze with statistical software A 10000 g E E Selected E 1 out shRNAs Oo O New Targets Untreated Control Cells 888 266 5066 Toll Free 650 968 2200 outside US Page 25 System Biosciences SBI User Manual E Recovering the shRNA templates from selected cells In order to identify sShRNAs from selected target cells with a specific phenotypic trait you will need to amplify and label shRNA targets with biotin for detection when hybridizing to Affymetrix Arrays The shRNA template inserts can be amplified from either genomic DNA or from RNA Isolation of RNA please refer to Appendix F for the protocol for starting with genomic DNA e In addition to isolating RNA from your samples you can also isolate RNA from non transduced target cells This RNA can be used as a negative con
76. transduce at least 2x10 target cells when using the Human 50K shRNA Library which has a complexity of about 200 000 cloned shRNA templates The following data show that infecting less than the recommended amount of cells results in loss of representation of shRNA constructs when comparing duplicate populations of infected cells 1 x 106 ifu amp 1 x 105 ifu amp 2 x 106 ifu amp 1 x 106 cells 1 x 105 cells 2 x 106 cells 30 infection efficiency 30 infection efficiency 50 infection efficiency 10 000 10 000 10 000 v v 1 000 amp 1 000 amp 1 000 3 8 sample 1 100 100 1 000 10 000 100 1 000 10 000 100 1 000 10 000 sample 2 sample 2 sample 2 Page 22 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 You should also consider that if more than 50 of target cells are infected by the shRNA library some infected cells will express more than one shRNA construct and may therefore knock down more than one gene simultaneously Day 1 1 Plate target cells in about six 6 10 cm plates at a density of about 5x10 cells per plate 24 hours prior to viral infection The optimal density of seeding should be adjusted in order to have about 50 confluency level with about 1x10 cells per plate at the time of infection Day 2 Add 10 ml of complete optimal medium with serum and antibiotics per plate and incubate cells at 37 C with 5 CO overnight Day 2 2 Quickly thaw the GeneNet shRN
77. trol for the amplification labeling and hybridization e Optional You can simultaneously isolate total genomic DNA to verify data generated by the total RNA and to measure copy number in the transduced cells 1 For each fraction of selected and reference cells detach cells from plates collect by centrifugation and wash in PBS Follow standard protocols for purification of total RNA and DNA from cells For most cell lines and tissue samples we recommend using TRIzol Reagent Invitrogen Cat 15596 026 DNase treatment of RNA samples is not necessary for the follow up protocol 2 After isolating total RNA measure the concentration e g by measuring absorbance at 260 nm and examine the integrity of the RNA by electrophoresis of a sample on a denaturing formaldehyde agarose EtBr gel or by using a BioAnalyzer Agilent Technologies High quality total RNA samples should appear as two bright ribosomal RNA bands at approximately 4 5 and 1 9 kb and at a ratio of about 2 1 Lower ratios are indicative of degradation Reverse Transcribe and Amplify Biotin labeled shRNA Targets Lentiviral constructs integrated into genomic DNA produce an alternative transcript from the CMV promoter that is a fusion of the marker gene copGFP or Puro with the shRNA sequence This alternative transcript is used as a template to amplify the shRNA insert Amplification of the inserts from total RNA requires two rounds of PCR During the second round of PCR with
78. ts Viral Titer arbitrary units MOLT 4 THP 1 human acute lymphoblastic leukemia human acute monocytic leukemia 100 100 80 80 779 FIV based pSIF1 copGFP HIV based pSIH1 copGFP 2 2 g 60 60 E E Q 40 Q 40 E 3 20 7 amp EFIV based pSIF1 copGFP X 20 HIV based pSIH1 copGFP 0 0 0 1 2 3 4 5 6 7 8 9 10 f 12 0 1 2 8 4 5 6 7 8 9 10 11 12 Viral Titer arbitrary units Viral Titer arbitrary units Page 44 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 Human Primary Stem Cell Lines PBMC donor HUVEC 3 passages donor peripheral blood mononuclear cells human umbilical vein endothelial cells 100 4 100 80 FIV based pSIF1 copGFP 80 ll HIV based pSIH1 copGFP m n g 60 g 60 i ig Q 40 9 40 2 2 X 20 aL 20 FIV based pSIF1 copGFP ll HIV based pSIH1 copGFP west tt 0 0 1 2 3 4 5 6 7 8 9 10 012 3 4 5 6 7 8 9 10 11 12 13 14 15 Viral Titer arbitrary units Viral Titer arbitrary units bone marrow human mesenchymal AML donor ind s
79. us step Page 28 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 a In the first round PCR Amplification prepare enough First Round PCR Master Mix for all reaction tubes plus one additional tube Combine the following components in the order shown Per Tube 72 yl Deionized H2O 10 ul 10X Titanium Tag PCR buffer 2 ul 50XdNTP mix 10 mM of each dNTP 2 wl Fwd GNF GNH Forward PCR Primer 10 uM 2 wl Rev GNF GNH Reverse PCR Primer 10 uM 2u 50X Titanium Taq DNA polymerase Do not use any H alternatives 90 ul Total volume b Mix contents by vortexing and spin the tube briefly in a microcentrifuge c Aliquot 90 ul of the PCR Master Mix into each tube from Step 1 j and place them in the hot lid thermal cycler Total volume should now be 100 pl with cDNA added d Commence thermal cycling using the following program 94 C for 2 min 94 C for 30 sec 68 C for 1 min 18 cycles 68 C for 3 min 15 C hold e When the program is complete analyze a 5 ul sample from each tube alongside a 50 bp DNA size marker by running on a 2 5 agarose EtBr gel in 1X TAE Note This will leave 95 ul of Master Mix remaining Compare your results to those below to confirm that your reactions were successful f Aliquot 1 ul from each tube into at least six new 0 5 ml reaction tubes You will need about 6 PCR reactions per sample to obtain enough biotin labeled shRNA target about 10 ug repeat previous s
80. vectors concentration to a very high titer and efficient gene transfer into mammalian and non mammalian cells Proc Natl Acad Sci USA 90 8033 8034 Cann A J ed 2000 RNA Viruses A Practical Approach Oxford Univ Press Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Naldini L 1998 A third generation lentivirus vector with a conditional packaging system J Virol 72 8463 8471 Gould D J and Favorov P 2003 Vectors for the treatment of autoimmune diseases Gene Therapy 10 912 927 Khvorova A Reynolds A and Jayasena D 2003 Functional shRNAs and miRNAs exhibit strand bias Cell 115 209 216 505 Lee N S Dohjima T Bauer G Li H Li M J Ehsani A Salvaterra P and Rossi J 2002 Expression of small interfering RNAs targeted against HIV 1 rev transcripts in human cells Nature Biotechnol 20 500 505 Lockhart D Dong H Byrne M C Follettie M T Gallo M V Chee M C Mittmann M Wang C Kobayashi M Horton H and Brown E L 1996 Expression monitoring by hybridization to high density oligonucleotide arrays Nat Biotech 14 1675 1680 Lorens J B Sousa C Bennett M K Molineaux S M and Payan D G The use of retroviruses as pharmaceutical tools for target discovery and validation in the field of functional genomics Curr Opin In Biotechnol 2001 12 613 621 Page 38 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XX
81. y knock down a target gene are summarized in the table below It is interesting to note that these criteria are very similar to those used to predict the most efficient short hybridization probes for microarrays Lokhard 1996 Perhaps this similarity is not surprising since both siRNA and expression profiling technologies are based on hybridization of antisense oligonucleotides target or antisense strand of siRNA with the complementary sequence in MRNA or the probe sequence immobilized on the array Criteria siRNA Sequence GeneChip Probes Size nucleotides 19 29 25 Homolog Unique lt 70 Unique lt 70 GC Content 40 50 40 60 Specific Sequences lt 4A T lt 6A T lt 5G lt 5G Secondary lt 0 kcal mol 0 kcal mol Structure Other GC rich 5 Yes based on AT rich 3 ends in target experimental data mRNA To take advantage of this finding we designed shRNA template sequences for our GeneNet shRNA Libraries that based on known parameters should work well to silence the targeted genes as well as hybridize to Affymetrix GeneChip Arrays When we tested siRNA constructs expressing sequences targeting p53 p73 and CD71 genes and designed to hybridize to Affymetrix arrays we found that at least 5096 of these Page 6 ver 5 080511 GeneNet Lentiviral shRNA Libraries Cat s SI2XXB 1 SIGXXB 1 siRNAs could efficiently silence the target mRNAs i e reduce expression by more than 70 These data confirm that GeneChip p

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