pdsipher and pdsipher -GFP shrna Vector User s Guide

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1 pdsipher and pdsipher -GFP shrna Vector User s Guide NOTE: PLEASE READ THE ENTIRE PROTOCOL CAREFULLY BEFORE USE Page 1. Introduction Vector Overview Vector Maps 2 4. Materials Provided Additional Materials and Equipment Required Cloning shrna Template Into pdsipher Vectors Transfection Protocol Selection of Plasmid Using Neomycin Technical Support Introduction The plasmid pdsipher is a 3.2 kb vector designed for expression of short hairpin RNA (shrna) in cultured cells following transfection. This shrna is approximately 60 nt long and is designed to form a hairpin structure that mimics 21 nt double stranded small interfering RNA (sirna) that induces RNA interference (RNAi) in gene knockdown experiments. The shrna is expressed from a human H1 promoter by endogenous polymerase III, resulting in high levels of expression of the shrna with a well-defined 3' end resulting in a UU overhang compatible with sirna design. Following successful transfection and expression of the shrna, it is processed by Dicer, the nuclease responsible for producing endogenous sirnas, and is processed into a form recognizable by the complex molecular machinery responsible for RNAi, known as the RISC complex. If the sirna has been designed with sufficient care, the RISC complex will then efficiently degrade RNA corresponding to the designed shrna. To design sirna and shrna sequences for your particular gene we recommend MoleculA s target design services (see for further details). 2 Vector Overview The plasmid pdsipher is 3.2 kb in size and contains many features suitable for shrna cloning, transfection and expression. A human H1 polymerase III promoter for efficient expression of the ~60 bp shrna with BamHI/HindIII cloning sites (bases ) for easy cloning of shrna constructs. Kanamycin/Neomycin dual resistance for both bacterial and mammalian selection (bases ). In addition, pdsipher -GFP contains a green fluorescent protein (GFP) under the control of a CMV promoter so cell transfection efficiencies can easily be assayed. MoleculA pdsipher technical manual January 2005

2 3 Vector Maps pdsipher (cat. #RI-500) Features Feature Position F1 origin SV40/Amp promoter Kan/Neo resistance TK p(a) site puc origin H1 pol. III promoter pdsipher -GFP (cat. #RI-550) Features Feature Position F1 origin SV40/Amp promoter Kan/Neo resistance TK p(a) site puc origin SV40 pa site GFP CMV promoter H1 pol. III promoter For the full sequence of these vectors, please visit 4 Materials Provided 10 µg lyophilized plasmid DNA Technical manual Storage Conditions pdsipher should be stored at -20 o C. Properly stored, product is guaranteed for one year from date of receipt. Safety Warning: To the present knowledge of the supplier, the materials supplied (listed above) do not contain any hazardous ingredients. However, all materials may present some unknown hazard and should be handled with caution. Some of the additional materials required (listed below) may present hazards and it is the sole responsibility of the user to determine what these hazards are and to take appropriate action. MoleculA pdsipher technical manual January 2005

3 5 Additional Materials and Equipment Required The following additional materials are required in order to use pdsipher and pdsipher - GFP to express shrna within cells. Cloning of cassette into pdsipher /pdsipher -GFP: BamHI HindIII Competent cells Annealed oligos corresponding to the shrna transcription cassette (see below for more details). Transfection of plasmid into cells: Cells at approximately 50-70% confluency Serum-Free Media Fetal Bovine Serum (FBS) Maxfect (MoleculA cat #TR ), or other DNA transfection reagent 6 Cloning shrna Template Into pdsipher Vectors 1) Design your sirna sequence. Different sirna sequences will knock down your gene of interest to different levels, due to only a small fraction of the RNA target being accessible for binding and interaction by sirna. We recommend that you design your sirna sequence using MoleculA s target design services. 2) Design shrna inserts for cloning into pdsipher vectors. This needs the following features: BamHI overhang for ligation into the vector. 19 nt sense sirna sequence. Loop sequence. 19 nt antisense sirna sequence. A six (6) T nt polymerase III termination sequence. HindIII overhang for ligation into the pdsipher vector. To design oligos with these features, please visit for our free shrna oligo design tool. 5' GATCC 3' G antisense TTCAAGAGA antisense TTTTTTA 3' antisense AAGTTCTCT antisense AAAAAATTCGA 5' 3) Order two oligos matching the above sequences. PAGE purification is recommended, however, many successful experiments have used standard desalted oligos. 4) Anneal the oligos. Set up the following reaction: Top-strand oligo (1 µg/µl) 1 µl Bottom-strand oligo (1 µg/µl) 1 µl 20X SSC 1 µl dh 2 O 17 µl Heat the reaction (95 C, 10 min) and place at room temperature (60 min). 5) Cut the vector with BamHI and HindIII. Run on a 1% agarose gel and gel purify the band. 6) Ligate the insert and vector together using standard DNA ligase protocols. 7) Transform the ligation into competent cells using protocols for that competent cell line. 8) Check that plasmids resulting from any colonies contain the insert, either by PCR or by restriction digest of the resulting plasmid(s). MoleculA pdsipher technical manual January 2005

4 7 Transfection Protocol - Using Maxfect For each cell line the protocol should be optimized according to the instructions shipped with Maxfect. Please ensure your transfection protocol is suitable for your cell line. The protocol listed here is for 6 well culture plate using Maxfect and should be optimized depending on cell type, well size, and transfection reagent. Although the protocol given here is for Maxfect, other lipid-based transfection reagents can be used with their respective protocols. 1) In a six well or 35mm tissue culture plate, seed ~2*10 5 cells per well in 2ml of D-MEM with 10%FBS. Incubate the cells at 37 C in a CO2 incubator until the cells are 50-70% confluent. This will usually take h. 2) Prepare the following solutions: Solution A: For each transfection, dilute 1µg pdsipher vector into 100µl serum-free or low protein medium without antibiotics. Solution B: For each transfection, dilute 2-8µl of lipid reagent into 100µl serum-free or low protein medium without antibiotics. Peak activity should be at about 6µl. 3) Add the pdsipher vector solution directly to the dilute lipid using a pipet. Mix gently by pipeting the solution and incubate the mixture 20 minutes at room temperature. 4) Wash the cells once with 2ml of low protein or serum-free and antibiotics free medium. 5) For each transfection, add 0.8ml of low protein or serum-free and antibiotics free medium to each tube containing the vector:lipid complex. Mix gently and overlay the diluted complex solution onto the washed cells. 6) Incubate the cells 5-7 h at 37 C in a CO2 incubator. 7) Add 1ml of medium containing 2 times the normal serum and antibiotics concentration (2x medium) without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium. 8) Incubate the cells an additional h. 9) Aspirate the medium and replace with fresh 1x medium containing the additives normally used to culture the cells. 10) Assay the cells using the appropriate protocol h after the addition of fresh medium in step 10 For the use of other transfection reagents, please follow the manufacturers' recommended protocol. MoleculA pdsipher technical manual January 2005

5 8 Selection of Plasmid Using Neomycin There are two main benefits for taking this approach: For difficult to transfect cell types, using neomycin selection will remove those cells that have not been transfected, leaving those cells that have successfully taken up the plasmid DNA. A stable cell line can be established, resulting in long-term shrna expression and target gene knockdown. To achieve this, the following protocol should be followed: 1) Perform a transient transfection as detailed above to assay the level of knockdown. 2) Following the transfection procedures detailed above, 24 hours post-transfection lift the cells from the plates using trypsin. Add G418 at a concentration of between 50 and 1500 µg/ml µg/ml is a good starting point. 3) Examine the wells every 48 hours to look for viable cells. Choose the lowest concentration of G418 that results in widespread cell death after 7 to 9 days, and kills all wild-type cells after 14 days. Use this concentration to select for resistant cell lines. Note: If your target gene is essential for cell viability, a stable cell line may not be obtained. 4) Linearizing the vector prior to transfection to aid integration into the host genome can increase the efficiency of the establishment of a stable cell line. Linearization should be performed with a restriction enzyme that does not cut in any of the vector's functional cassettes (e.g. The HI promoter or the GFP expression cassette) such as MluI. 9 Technical Support For technical assistance please contact us at MoleculA pdsipher technical manual January 2005

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