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Long-Term Gene Silencing in Mammalian Cells Using Sirna

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Long-Term Gene Silencing in Mammalian Cells Using Sirna 1032295_0805_SPOS_ELSO_Meeting.qxd 12.08.2005 18:29 Uhr Seite 1 Long-term gene silencing in mammalian cells using siRNA Frank Narz, Silke Janhsen, and Martin Weber QIAGEN GmbH, Hilden, Germany Introduction Summary of results Gene silencing using siRNA-mediated RNAi has become a powerful tool in cell biology, addressing a range of The newly developed transfection protocol allows gene knockdown in mammalian cells for over 2 weeks. research questions in functional genomics and drug discovery. After delivery of siRNA into the cell, rapid degradation of the target mRNA can be detected, often within 24 hours, and subsequently the corresponding The protocol involves siRNA delivery using HiPerFect Transfection Reagent every time the cells are diluted and protein is also knocked down. replated. This knockdown is transient, especially when working with proliferating cell lines where the intracellular siRNA is The low cytotoxicity of HiPerFect Transfection Reagent, in combination with its suitability for transfection of low diluted with each cell division. As most cell lines have to be subcultured approximately twice a week, siRNA amounts, allows repeated siRNA transfection without affecting cell viability. siRNA-mediated RNAi experiments are usually analyzed 3–4 days after transfection. In many cases, this Data presented here show long-term silencing of MAPK1 and lamin A/C in HeLa and MCF-7 cells using short-term knockdown is too brief and phenotypic effects that require a longer duration of knockdown of the various siRNA concentrations (Figures 2 and 4). target protein cannot be examined. Viability staining showed that cells were healthy after repeated transfections when HiPerFect Transfection We have developed a protocol that allows gene silencing in mammalian cells for more than 2 weeks. siRNA is Reagent was used (Figures 3 and 5). transfected using lipid-based HiPerFect Transfection Reagent every time the cells are diluted and plated into a new culture plate. A phenotypic assay analyzing melanin synthesis and secretion after tyrosinase knockdown identifies this pathway as a model system where long-term silencing is necessary for melanin research (Figures 6, 7, and 8). Trademarks: QIAGEN®, GeneGlobe™, QuantiTect®, RNeasy® (QIAGEN Group); TaqMan® (Roche Group). siRNA technology licensed to QIAGEN is covered by various patent applications, owned by the Massachusetts Institute of Technology, Cambridge, MA, USA and others. Purchase of QIAGEN products for PCR containing HotStarTaq DNA Polymerase is accompanied by a limited license to use them in the polymerase chain reaction (PCR) process for research and development activities in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as purchased, i.e. an authorized thermal cycler. The PCR process is covered by the foreign counterparts of U.S. Patents Nos. 4,683,202 and 4,683,195 owned by F. Hoffmann-La Roche Ltd. The 5' nuclease process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. © 2005 QIAGEN, all rights reserved. RNAi Leads to Transient Knockdown Protocol for long-term silencing using HiPerFect Reagent When siRNA is delivered into the cell, it meditates RNAi Mechanism Addition of siRNA–HiPerFect Reagent complexes after cells are split allows long-term knockdown. The detailed cleavage of homologous mRNA and so translation protocol is available from QIAGEN Technical Services. is prevented (see flowchart). Synthetic siRNA (21–23 nt dsRNA) Long-Term Gene Silencing Procedure mRNA degradation leads to knockdown of the RISC Assembly target protein. However the effect is transient and gene expression is restored as siRNAs are diluted Prepare complexes with each cell division and if cell cultures are split (Figure 1). RNA unwound single-stranded siRNA incorporated into RISC Apply to cells Transient mRNA and Protein Knockdown Incubate Change medium after 6–24 h Transfection mRNA target When cells are confluent: recognition Remove medium Wash cells Add Trypsin/EDTA Protein RNA Add medium Prepare complexes mRNA cleavage Transfer to a new plate ~48h ~96h ~120h Figure 1 siRNA transfection leads to mRNA and subsequently protein knockdown, Add complexes but the effects are short-lived. Incubate Change medium after 6–24 h Long-term silencing in HeLa and MCF-7 cells Long-term silencing does not affect cell viability Transfection using 5 nM MAPK1 siRNA and HiPerFect Transfection Reagent each time cells were split resulted Cell viability was assessed by staining cells with Calcein AM dye after either a single transfection or after in prolonged knockdown of the target mRNA (Figure 2). 3 transfections. Calcein AM fluoresces green in living cells. When cells were transfected only once, repeated splitting cycles resulted in loss of the silencing effect (Figure 2). Calcein AM staining shows that repeated siRNA transfection using HiPerFect Transfection Reagent does not affect cell viability (Figure 3). Long-term MAPK1 silencing in HeLa cells resulted in reduced cell numbers, probably caused by reduced proliferation. Effective MAPK1 Silencing after Multiple Splitting Cycles The same samples were used for gene expression analysis and results showed that repeated transfections A HeLa B MCF-7 resulted in prolonged silencing (Figure 4). 140 250 120 200 100 Viable Cells after Repeated Transfections 150 80 60 100 Untransfected Mock MAPK1 siRNA GFP siRNA Lamin A/C siRNA 40 transfected Relative expression Relative expression 50 of MAPK1 mRNA (%) of MAPK1 mRNA (%) 20 0 0 One initial One One transfection One initial transfection One initial transfection transfection Transfection transfection Transfection Untransfected Untransfected Untransfected Untransfected with every split with every split Not split Split 5 times Not split Split 5 times 3 consecutive transfections Figure 2 A HeLa or B MCF-7 cells (8 x 104) were plated in 24-well plates and transfected with 5 nM MAPK1 siRNA. After 48 hours, control cultures that had not been split were lysed and RNA was purified using the RNeasy® Mini Kit or the RNeasy 96 Kit, followed by gene expression analysis using the gene-specific TaqMan® probe and primers and the QuantiTect® Probe RT-PCR Kit. The expression level of MAPK1 was normalized to that of GAPDH. The remaining cultures were split twice a week and either retransfected at each split or replated without transfection. After 18 days, MAPK1 expression was determined by quantitative, real-time RT-PCR as described. Figure 3 HeLa cells (8 x 104) were plated in 24-well plates and transfected with 20 nM siRNA targeting MAPK1, GFP (nonsilencing siRNA), or lamin A/C. Mock transfected cells (HiPerFect Reagent only) were also examined. Cultures were split twice a week and either retransfected at each split or replated without transfection. After 9 days and 2 splitting cycles, viable cells were stained using Calcein AM. These cells were also used for gene expression analysis by quantitative, real-time RT-PCR (see Figure 4). Repeated transfection prolongs silencing without affecting Choice of transfection reagent affects viability viability HeLa cells were transfected with MAPK1 siRNA Cell Viability Using Different Transfection Reagents using HiPerFect Transfection Reagent, Reagent L, or Repeated siRNA transfection using HiPerFect The viability of the cells in these experiments was not Reagent O. The siRNA concentrations used were HiPerFect Reagent L Reagent O Reagent results in prolonged silencing (Figure 4). affected by repeated siRNA transfection (Figure 3). based on the manufacturers’ instructions: 5 nM with One transfection HiPerFect Reagent; 33 nM with Reagent L; 106 nM Split twice with Reagent O (Figure 5). Prolonged Knockdown after Repeated Transfections HiPerFect Reagent allowed the use of the lowest Three transfections A B siRNA concentration (5 nM) and cells were healthy Split twice Untransfected GFP siRNA Mock transfected MAPK1 siRNA and viable after transfection. Untransfected GFP siRNA Lamin A/C siRNA 160 200 Figure 5 HeLa cells (8 x 104) were plated in 24-well plates and transfected using 160 Reagent L required the use of a higher siRNA con- 120 5 nM MAPK1 siRNA and HiPerFect Reagent, 33 nM MAPK1siRNA and 120 centration (33 nM) and toxicity was evident after Reagent L, or 106 nM MAPK1 siRNA and Reagent O. siRNA concentrations 80 were recommended by the manufacturer. Cultures were split twice a week and 80 repeated transfection. Cells transfected with Reagent either retransfected at each split or replated without transfection. After 9 days, 40 40 O did not show cytotoxicity, but this reagent cell viability was examined by phase contrast microscopy. Relative expression Relative expression of MAPK1 mRNA (%) 0 of lamin A/C mRNA (%) 0 required the use of the highest siRNA concentration One transfection One initial Transfection with One transfection One initial Transfection with (106 nM). transfection every split transfection every split Not split Split twice Not split Split twice Figure 4 HeLa cells were transfected with 20 nM siRNA targeting A MAPK1 orB lamin A/C. siRNA targeting GFP (nonsilencing siRNA) was also transfected. Mock transfected cells were also analyzed. After 48 hours, control cultures that had not been split were used for quantitative, real-time RT-PCR analysis. Cultures were split twice a week and either retransfected at each split or replated without transfection. After 9 days and 2 splitting cycles, MAPK1 and lamin A/C expression was determined by quantitative, real-time RT-PCR. Cell viability of these cultures was also assessed (Figure 3). Melanin synthesis is a candidate model system requiring Silencing of the melanin synthesis key enzyme tyrosinase long-term silencing B16-F1 cells were transfected with each one of Tyrosinase knockdown was analyzed by western 2 HP GenomeWide siRNAs targeting tyrosinase or blotting. After 72 hours, tyrosinase was effectively B16-F1 is a murine melanocytic cell line that B16-F1 Cells Secrete Melanin into the Medium with lamin A/C siRNA as a negative control. knocked down at the protein level using each synthesizes melanin.
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