《pLKO.1 – TRC Cloning Vector-protocol.docx》由会员分享，可在线阅读，更多相关《pLKO.1 – TRC Cloning Vector-protocol.docx（15页珍藏版）》请在taowenge.com淘文阁网|工程机械CAD图纸|机械工程制图|CAD装配图下载|SolidWorks_CaTia_CAD_UG_PROE_设计图分享下载上搜索。
1、pLKO.l - TRC Cloning VectorAddgene Plasmid 10878. Protocol Version 1.0. December 2006.Copyright Addgene 2006, All Rights Reserved. This protocol is provided for your convenience. See “warranty information in appendix.Table of ContentsA. pLKO.l-TRC Cloning Veclor A.l The RNAi ConsortiumA.2 Map ofpLKO
2、.l A.3 Related plasmidsB. Designing shRNA Oligos for pLKO.l B.l Determine the optimal 21 -mer targets in your geneB.2 Order oligos compatible with pLKO.l C. Cloning shRNA oligos inlo pLKO.lC.l Recommended materials C.2 Annealing oligosC.3 Digesting pLKO.l TRC-Cloning Vector C.4 Ligating and transfor
3、ming into bacteriaD. Screening for Inserls D.l Recommended materialsD.2 Screening fbr inserts E. Producing Lcntiviral ParticlesE.l Recommended materials E.2 Protocol for producing lentiviral particlesF. Infecting Targel Cells F.l Recommended materialsF.2 Determining the optimal puromycin concentrati
4、on F.3 Protocol fbr lentiviral infection and selectionG. Safety H. ReferencesH.l Published articles H.2 Web resourcesI. Appendix 1.1 Sequence of pLKO. I TRC-Cloning Vector1.2 Recipes 1.3 Warranty information You may want to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to
5、obtain the ratio that gives you the optimal viral production.d. Create a master mix of FuGENE 6 transfection reagent in serum-free OPTI-MEM. Calculate the amount of Fugcne and OPTI-MEMnccessary given that each reaction will require 6 pL FuGENE + 74 pL OPTI-MEM. For example: lx master mix: 6 jiL FuGE
6、NE + 74 pL OPTI-MEM5x master mix: 30 gL FuGENE + 370 pL OPTI-MEM lOx master mix: 60 pL FuGENE + 740 pL OPTI-MEMIn a polypropylene tube, add OPTI-MEM first. Pipette FuGENE directly into(he OPTI-MEM - do not allow FuGENE to come in contact with the wails of the (ubebefore it has been diluted. Mix by s
7、wirling or gently flicking the tube. Incubate for 5 minutes at room temperature.e. Add 80 pL of FuGENE master mix to each tube from step c for a total volume of 100 pL. Pipette master mix directly into the liquid and not onto the walls of the tube. Mix by swirling or gently flicking the tube.f. Incu
8、bate for 20-30 minutes at room temperature.g. Retrieve HEK-293T cells from incubator. The cells should be 50-80% confluent and in DM EM that does not contain antibiotics.h. Without touching the sides of the dish, gently add DNA:FuGENE mix dropwise to cells. Swirl to disperse mixture evenly. Do not p
9、ipette or swirl too vigorously, as you do not want to dislodge the cells from the plate.i. Incubate cells at 37, 5% CO2 for 12-15 hours.Day 3:j. In the morning, change the media to remove the transfection reagent. Replace with 5 mL fresh DM EM + 10% FBS + penicillin/streptomycin. Pipette the media o
10、nto the side of the plate so as not to disturb the transfected cells.k. Incubate cells at 37, 5% CO2 for 24 hours.Day 4:l. Harvest media from cells and transfer to a polypropylene storage tube. The media contains your lentiviral particles. Store at 4.m. Add 5 mL of fresh media containing antibiotics
11、 to the cells and incubate at 37, 5% CO2 for 24 hours.Day 5:n. Harvest media from cells and pool with media from Day 4. Spin media at 1,250 rpm for 5 minutes to pellet any HEK-293T cells that were inadvertently collected during harvesting. In lieu of centrifiigation, you may filter the media through
12、 a 0.45 pm filter to remove the cells. Do not use a 0.2 pm filter, as this is likely to shear the envelope of your virus.o. Virus may be stored at 4 for a few days, but should be frozen at -20 or -80 for long-term storage. Freeze/thaw cycles decrease the efficiency of the virus, so Addgene recommend
13、s that you use the virus immediately or aliquot the media into smaller tubes to prevent multiple freeze/thaw cycles.F. Infecting Target CellsLentiviral particles can efficiently infect a broad range of cell types, including both dividing and non-dividing cells. Addition of puromycin will allow you t
14、o select for cells that are stably expressing your shRNA of interest.F.l. Recommended MaterialsMaterialVendor and catalog #Hexadimethrine bromide (polybrene)*Sigma-Aldrich: #H9268Protamine Sulfate*MP Biomcdicals: #194729Puromycin*Sigma-Aldrich: #P8833Target cellsVaries based on your experimerCulture
15、 media for target cellsVaries based on your experimerMaterials for assayVaries based on your experimer Detailed protocols for preparing polybrene, protamine sulfate, and puromycin are located in the Appendix”.F.2. Determining the Optimal Puromycin ConcentrationEach cell line responds differently to
16、puromycin selection. Addgene strongly recommends that you determine the optimal puromycin concentration for your cell line before initiating your experiment.Day 1:a. Plate target cells in ten 6 cm plates and grow at 37 C, 5% CO2 overnight.Day 2:b. The target cells should be approximately 80-90% conf
17、luent.c. Dilute puromycin in the preferred culture media for your target cells. The final concentration of puromycin should be from 1-10 pg/mL in I pg/mL increments.d. Label plates from 1-10 and add appropriate puromycin-containing media to cells.Days 3+:e. Examine cells each day and change to fresh
18、 puromycin-containing media every other day.f. The minimum concentration of puromycin (hat results in complete cell death after 3-5 days is the concentration that should be used for selection in your experiments. (You may wish to repeat this titration with finer increments of puromycin to determine
19、a more precise optimal puromycin concentration.)F.3. Protocol for Lentiviral Infection and SelectionDay 1:a. Plate target cells and incubate at 37, 5% CO2 overnight.Day 2:b. Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 pg/mL polybrene. Polybrene incr
20、eases the efficiency of viral infection. However, polybrene is toxic to some cell lines. In these cell lines, substitute protamine sulfate for polybrene.c. Add lentiviral particle solution from step E. For a 6 cm target plate, add between 0.05-1 mL virus (add 0.5 mL for a high MOI, and 3 days 4 days
21、 4 daysAssaymRNA knockdown (quantitative PCR)Prolein knockdown (western blot)Phenotypic assayG. SafetyBL2 safety practices should be followed when preparing and handling lentiviral particles. Personal protective clothing should be worn at all times. Use plastic pipettes in place of glass pipettes or
22、 needles. Liquid waste should be decontaminated with at least 10% bleach.Laboratory materials that come in contact with viral particles should be treated as biohazardous waste and autoclaved. Please follow all safety guidelines from your institution and from the CDC and NIH for work in a BL2 facilit
23、y.If you have any questions about what safety practice to follow, please contact your institutions safety office.To obtain (he MSDS for this product, visit and follow the MSDS link.H. ReferencesH.l. Published ArticlesKlivorova A ct. al. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 11
24、5:209-216. (PubMed)Moffat J ct. al. 2006. A Icntiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283-1298. (PubMed)Naldini L et. al. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:26
25、3-267. (PubMed)Schwarz DS et. al. 2003. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199-208. (PubMed)Stewart SA et. al. 2003. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9(4):493-501. (PubMed)Zufferey R et. al. 1997. Multiply attenuated lentiviral vect
26、or achieves efficient gene delivery in vivo. Nat Biotechnol 15(9):871-5. (PubMed)Zufferey R et. al. 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873-80. (PubMed)H.2. Web resourcesAddgcncs mammalian RNAi website: The RNAi Consortium (TRC): Ba
27、ckground on RNAimechanism: Whitehead siRNA Selection Program: Mekentosj iRNAi Program: Back lo Top1. Appendix1.1. Sequence of pLKO.l TRC-Cloning VectorClick here to see the sequence of pLKO.l TRC-cloning vector. The vector is 8901 base pairs total, and the stuffcr insert is shown in all capital lett
28、ers.1.2. RecipesLuria Broth Agar (LB agar) + antibioticPer 40 grams of powder from American Bioanalytical catalog # ABO 1200-02000, LB contains:1 Og tryptone5g yeast extract10g sodium chloride15g agar Prepare LB agar solution by dissolving 40g of LB powder in IL of distilled water. Autoclave and coo
29、l to 55. Add 1mL of 1 OOmg/mL ampicillin or carbenicillin to obtain a final concentration of 100 pg/mL antibiotic. Pour plates and store at 4.Hexadiniethrine Bromide (Polybrene)Prepare a 1 mg/mL solution of polybrene (Sigma-Aldrich catalog #H9268) in 0.9% NaCl. Autoclave to sterilize. Stock solution
30、 is stable at 4 for up to one year. The powder form of polybrene is stable at 4 for several years.Protamine SulfateStore protamine sulfate (MP Biomedicals catalog #194729) at 4. Freely soluble in hot water and slightly soluble in cold water.PuromycinPrepare a 50ing/mL stock solution of puromycin (Si
31、gma-Aldrich catalog #P8833) in distilled water. Sterilize by passing through a 0.22 pm filter. Store aliquots at -20.1.3. Warranty InformationAddgene is committed to providing scientists with high-quality goods and services. Addgene makes every eflbrl to ensure the accuracy of its literature, bul re
32、alizes that typographical or other errors may occur. Addgene makes no warranty of any kind regarding the contents of any literature. Literature are provided to you as a guide and on an “AS IS” “AS AVAILABLE basis without warranty of any kind either expressed or implied, including but not limited to
33、the implied warranties of fitness for a particular purpose, non-infringement, typicality, safety and accuracy.The distribution of any literature by Addgene is not meant to carry with i(, and does not grant any license or rights of access or use to the materials described in the literature.The distri
34、bution of materials by Addgene is not meant to carry with it, and does not grant any license, express or implied, under any patent. All transfers of materials from Addgcnc to any party are governed by Addgenes Terms of Use, Addgenes Terms of Purchase, and applicable Material Transfer Agreements betw
35、een the party that deposited the material at Addgene and the party receiving the material.A. pLKO. 1 -TRC Cloning VectorA.l The RNAi ConsortiumThe pLKO.l cloning vector is the backbone upon which The RNAi Consortium has built a library of shRNAs directed against 15,000 human and 15,000 mouse genes.
36、Addgene is working with the TRC to make this shRNA cloning vector available to the scientific community. Please cite Moffat et aL, Cell 2006 Mar; 124(6):1283-98 (PubMed,) in all publications arising from the use of this vector.A.2 Map ofpLKO.lpLKO. 1 is a replication-incompetent lentiviral vector ch
37、osen by the TRC for expression of shRNAs. pLKO.l can be introduced into cells via direct transfection, or can be converted into lentiviral particles for subsequent infection of a target cell line. Once introduced, the puromycin resistance marker encoded in pLKO.l allows for convenient stable selecti
38、on.shRNA ConstructAmpRFigure 1 : Map of pLKO.l containing an shRNA insert. The original pLKO. 1-TRC cloning vector has a 1.9kb staffer that is released by digestion with A gel and EcoRI. shRNA oligos are cloned into the Agel and EcoRI sites in place of the stuffer. The Agel site is destroyed in most
39、 cases (depending on the target sequence), while the EcoRI site is preserved. For a complete map of pLKO.l containing the 1.9kb stuffen visit .Description Vector ElementU6Human U6 promoter drives RNA Polymerase III transcription for generation of shRNA transcripts.cPPTCentral polypurinc tract, cPPT,
40、 improves transduction efficiency by facilitating nuclear import of the the transduced cells.hPGKHuman phosphoglycerate kinase promoter drives expression of puromycin.Puro RPuromycin resistance gene for selection of pLKO.l plasmid in mammalian cells.sin 3LTR3 Sclf:inactivating long terminal repeat.f
41、l orifl bacterial origin of replication.Amp RAmpicillin resistance gene for selection of pLKO. 1 plasmid in bacterial cellspUC oripUC bacterial origin of replication.5LTR5 long terminal repeat.RRERev response element.7shRNAL IConstruct _7 sgm，)。p| 人也/ SCCGGxxxxxxxxxxxxxxxxxxxxxCTCGAGxxxxxxxxxxxxxxxx
42、xxxxxTTTTT-J GGCCxxxxxxxxxxxxxxxxxxxxxGAGCTCxxxxxxxxxxxxxxxxxxxxxAAAAA1 w1 tramcrlptionshRNAr JA.3 Related ProductsDescriptionThe following plasmids available from Addgene are recommended for use in conjunction with the pLKO.l TRC-cloning vector.Plasmid (Addgene ID #)dLKO.1 TRC controlNegative contr
43、ol vector containing non-hairpin insert.pLKO.l - scramble shRNANegative control vector containing scrambled shRNA.psPAX2Packaging plasmid for producing viral particles.pMD2.GEnvelope plasmid fbr producing viral particles.Note: pLKO.l can also be used with packaging plasmid pCMV-dR8.2 dvpr and envelo
44、pe plasmid pCMV-VSVG from Robert Weinbergs lab. For more information, visit Addgenes Mammalian RNAi Tools page.Several other laboratories have deposited pLKO derived vectors that may also be useful for your experiment. To see these vectors, visit Addgcncs website and “search fdr “pLKO”.B. Designing shRNA Oligos for pLKO. 12.1 Determining the Optimal 21-mer Targets in your GeneSelection