AM5770: pSilencer™ 3.1-H1 neo

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	CT26 murine colon carcinoma cells, syngeneic to BALB/C mice, were grown as monolayer cultures in DMEM–10% FBS (Invitrogen, Carlsbad, CA) supplemented with 100 IU/ml Penicillin and 100 µg/ml Streptomycin. Cells were maintained in a 37°C incubator with 5% CO 2 humidified air. Cell lines that were stably transfected with plasmid vectors were maintained in DMEM–10% FBS supplemented with 100 IU/ml Penicillin, 100 µg/ml Streptomycin and 750 µg/ml Geneticin (Invitrogen).Using GenBank™ sequence NM 009263 for murine OPN cDNA and computer analysis software developed by Ambion, Inc., we selected two candidate sequences in the OPN cDNA sequence for RNAi ( Figure 1A ). These 21-nt sequences showed no homology with other known mouse genes. The corresponding sense and antisense siRNA sequences for each target are also shown ( Figure 1A ). Synthetic, annealed, siRNA oligonucleotides were synthesized chemically and gel-electrophoresis purified (Ambion, Austin, TX) and used during transient transfection experiments. Murine mismatch or scrambled siRNA sequences (Ambion) possessing limited homology to mouse genes served as a negative control. For stable RNAi we designed a hairpin siRNA sequence that contains both sense and antisense siRNA sequences against OPN target 2 and flanking BamH1 and HindIII sites ( Figure 1E ). This sequence was chemically synthesized and PAGE-purified (Sigma-Genosys, The Woodlands, TX) and cloned into pSilencer neo™, an expression vector containing an H1 RNA polymerase III promoter ( 24 ) and a neomycin antibiotic resistance gene (Ambion). A pSilencer neo™ vector that expresses mismatch hairpin siRNA with limited homology to mouse genes (Ambion) served as our negative control. Harvesting of CT26 cells using Trypsin was done 24 h prior to transfection, and plated at a density of 5 × 10 5 cells/well in 6-well plates (Costar, Corning Inc., NY) in DMEM–10% FBS without antibiotics. Reconstituted, annealed siRNA against target 1 (sR1) and siRNA against target 2 (sR2) were diluted in OPTIMEM I (Invitrogen) to a final concentration of 50 nM and transiently transfected into CT26 using Lipofectamine 2000 (Invitrogen). The medium was replaced with DMEM–10% FBS after 4 h. CT26 wild-type cells (WT), CT26 incubated with Lipofectamine 2000 alone in the absence of siRNA and CT26 incubated with Lipofectamine 2000 and mismatch siRNA (sR−) were used as controls. Cells were harvested 48 h after transfection and OPN protein levels were quantified by western blot-analysis in triplicate assays.

Protocol tips

CT26 murine colon carcinoma cells, syngeneic to BALB/C mice, were grown as monolayer cultures in DMEM–10% FBS (Invitrogen, Carlsbad, CA) supplemented with 100 IU/ml Penicillin and 100 µg/ml Streptomycin. Cells were maintained in a 37°C incubator with 5% CO 2 humidified air. Cell lines that were stably transfected with plasmid vectors were maintained in DMEM–10% FBS supplemented with 100 IU/ml Penicillin, 100 µg/ml Streptomycin and 750 µg/ml Geneticin (Invitrogen).Using GenBank™ sequence NM 009263 for murine OPN cDNA and computer analysis software developed by Ambion, Inc., we selected two candidate sequences in the OPN cDNA sequence for RNAi ( Figure 1A ). These 21-nt sequences showed no homology with other known mouse genes. The corresponding sense and antisense siRNA sequences for each target are also shown ( Figure 1A ). Synthetic, annealed, siRNA oligonucleotides were synthesized chemically and gel-electrophoresis purified (Ambion, Austin, TX) and used during transient transfection experiments. Murine mismatch or scrambled siRNA sequences (Ambion) possessing limited homology to mouse genes served as a negative control. For stable RNAi we designed a hairpin siRNA sequence that contains both sense and antisense siRNA sequences against OPN target 2 and flanking BamH1 and HindIII sites ( Figure 1E ). This sequence was chemically synthesized and PAGE-purified (Sigma-Genosys, The Woodlands, TX) and cloned into pSilencer neo™, an expression vector containing an H1 RNA polymerase III promoter ( 24 ) and a neomycin antibiotic resistance gene (Ambion). A pSilencer neo™ vector that expresses mismatch hairpin siRNA with limited homology to mouse genes (Ambion) served as our negative control. Harvesting of CT26 cells using Trypsin was done 24 h prior to transfection, and plated at a density of 5 × 10 5 cells/well in 6-well plates (Costar, Corning Inc., NY) in DMEM–10% FBS without antibiotics. Reconstituted, annealed siRNA against target 1 (sR1) and siRNA against target 2 (sR2) were diluted in OPTIMEM I (Invitrogen) to a final concentration of 50 nM and transiently transfected into CT26 using Lipofectamine 2000 (Invitrogen). The medium was replaced with DMEM–10% FBS after 4 h. CT26 wild-type cells (WT), CT26 incubated with Lipofectamine 2000 alone in the absence of siRNA and CT26 incubated with Lipofectamine 2000 and mismatch siRNA (sR−) were used as controls. Cells were harvested 48 h after transfection and OPN protein levels were quantified by western blot-analysis in triplicate assays.

Publication protocol

CT26 murine colon carcinoma cells, syngeneic to BALB/C mice, were grown as monolayer cultures in DMEM–10% FBS (Invitrogen, Carlsbad, CA) supplemented with 100 IU/ml Penicillin and 100 µg/ml Streptomycin. Cells were maintained in a 37°C incubator with 5% CO 2 humidified air. Cell lines that were stably transfected with plasmid vectors were maintained in DMEM–10% FBS supplemented with 100 IU/ml Penicillin, 100 µg/ml Streptomycin and 750 µg/ml Geneticin (Invitrogen).Using GenBank™ sequence NM 009263 for murine OPN cDNA and computer analysis software developed by Ambion, Inc., we selected two candidate sequences in the OPN cDNA sequence for RNAi ( Figure 1A ). These 21-nt sequences showed no homology with other known mouse genes. The corresponding sense and antisense siRNA sequences for each target are also shown ( Figure 1A ). Synthetic, annealed, siRNA oligonucleotides were synthesized chemically and gel-electrophoresis purified (Ambion, Austin, TX) and used during transient transfection experiments. Murine mismatch or scrambled siRNA sequences (Ambion) possessing limited homology to mouse genes served as a negative control. For stable RNAi we designed a hairpin siRNA sequence that contains both sense and antisense siRNA sequences against OPN target 2 and flanking BamH1 and HindIII sites ( Figure 1E ). This sequence was chemically synthesized and PAGE-purified (Sigma-Genosys, The Woodlands, TX) and cloned into pSilencer neo™, an expression vector containing an H1 RNA polymerase III promoter ( 24 ) and a neomycin antibiotic resistance gene (Ambion). A pSilencer neo™ vector that expresses mismatch hairpin siRNA with limited homology to mouse genes (Ambion) served as our negative control. Harvesting of CT26 cells using Trypsin was done 24 h prior to transfection, and plated at a density of 5 × 10 5 cells/well in 6-well plates (Costar, Corning Inc., NY) in DMEM–10% FBS without antibiotics. Reconstituted, annealed siRNA against target 1 (sR1) and siRNA against target 2 (sR2) were diluted in OPTIMEM I (Invitrogen) to a final concentration of 50 nM and transiently transfected into CT26 using Lipofectamine 2000 (Invitrogen). The medium was replaced with DMEM–10% FBS after 4 h. CT26 wild-type cells (WT), CT26 incubated with Lipofectamine 2000 alone in the absence of siRNA and CT26 incubated with Lipofectamine 2000 and mismatch siRNA (sR−) were used as controls. Cells were harvested 48 h after transfection and OPN protein levels were quantified by western blot-analysis in triplicate assays.

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