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The inhibition of Syn G expression by siRNA was carried out by vector-based RNA interference approaches. pSUPER.retro.neo+gfp was used as a vector (Oligoengine, Inc., Seattle, WA). This retroviral vector ensures efficient siRNA expression using H1 RNA polymerase III promoter, which drives the endogenous production of siRNA. For oligonucleotide design the software from Dharmacon and Whitehead Institute were used. The designed oligonucleotides correspond to different parts of the rat Syn G gene, including exons 3 and 4 (E3 and E4) and 3′-untranslated region (3′-UTR) (Table 1). As a control, scrambled (Scr) nucleotide sequence corresponding to E3 was used. The scrambled sequence has the same nucleotide composition as the input sequence and does not possess a significant homology to other genes, according to BLAST analysis. Each double strand oligo contained the BglII site on 5′- and 3′-end on the HindIII. The oligonucleotides for candidate siRNAs were analyzed by BLAST search to exclude considerable similarity to other genes. Oligonucleotides were inserted into pSUPER vector by ligation using BglII and HindIII sites as recommended by the manufacturer (Oligoengine, Inc.). Further steps (annealing, linearization, cloning the annealed oligonucleotides into the vector, and transformation of Escherichia coli) were performed according to the manufacturer's protocols. The correctness of all constructs was confirmed by sequencing. On the next step, pSUPER.retro.plasmids were transfected into 293T cells together with a packaging plasmid pCLEco (Imgenex, San Diego, CA) using FUGENE-HD transfection reagent (Roche). Transfection into 293T cells was performed following the manufacturer's protocol. The cells (105/ml) were split into 60-mm plates with Dulbecco's modified Eagle's medium and grown overnight. Before transfection the media was changed to a fresh one. DNA was incubated with FUGENE-HD (ratio 3:2, 30 min, room temperature) to form a complex. Each mixture contained 1.5 μg of pSUPER-plasmid DNA and 0.5 μg of pCLeo DNA. After incubation the mixture was added to cells in the media containing fetal bovine serum without antibiotics. In 48 h the fluorescence was analyzed using the Bio-Rad Microradians Plus confocal system coupled to a Nikon Eclipse inverted microscope TE300 (Melville, NY). The efficiency of transfection was ∼50%. The media from the 293T cells containing the virus together with 10 μg/ml of Polybrene (Millipore Corporation, Billerica, MA) were used for the infection of RGC-5. After 24 h of growth the efficiency of infection determined as described above was ∼30%. Because pSUPER plasmid contained green fluorescent protein encoding nucleotide sequence, we used flow cytometry (BD-FACSAria Cell Sorting System, BD Biosciences) to enrich the population of cell containing siRNA. |
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| The inhibition of Syn G expression by siRNA was carried out by vector-based RNA interference approaches. pSUPER.retro.neo+gfp was used as a vector (Oligoengine, Inc., Seattle, WA). This retroviral vector ensures efficient siRNA expression using H1 RNA polymerase III promoter, which drives the endogenous production of siRNA. For oligonucleotide design the software from Dharmacon and Whitehead Institute were used. The designed oligonucleotides correspond to different parts of the rat Syn G gene, including exons 3 and 4 (E3 and E4) and 3′-untranslated region (3′-UTR) (Table 1). As a control, scrambled (Scr) nucleotide sequence corresponding to E3 was used. The scrambled sequence has the same nucleotide composition as the input sequence and does not possess a significant homology to other genes, according to BLAST analysis. Each double strand oligo contained the BglII site on 5′- and 3′-end on the HindIII. The oligonucleotides for candidate siRNAs were analyzed by BLAST search to exclude considerable similarity to other genes. Oligonucleotides were inserted into pSUPER vector by ligation using BglII and HindIII sites as recommended by the manufacturer (Oligoengine, Inc.). Further steps (annealing, linearization, cloning the annealed oligonucleotides into the vector, and transformation of Escherichia coli) were performed according to the manufacturer's protocols. The correctness of all constructs was confirmed by sequencing. On the next step, pSUPER.retro.plasmids were transfected into 293T cells together with a packaging plasmid pCLEco (Imgenex, San Diego, CA) using FUGENE-HD transfection reagent (Roche). Transfection into 293T cells was performed following the manufacturer's protocol. The cells (105/ml) were split into 60-mm plates with Dulbecco's modified Eagle's medium and grown overnight. Before transfection the media was changed to a fresh one. DNA was incubated with FUGENE-HD (ratio 3:2, 30 min, room temperature) to form a complex. Each mixture contained 1.5 μg of pSUPER-plasmid DNA and 0.5 μg of pCLeo DNA. After incubation the mixture was added to cells in the media containing fetal bovine serum without antibiotics. In 48 h the fluorescence was analyzed using the Bio-Rad Microradians Plus confocal system coupled to a Nikon Eclipse inverted microscope TE300 (Melville, NY). The efficiency of transfection was ∼50%. The media from the 293T cells containing the virus together with 10 μg/ml of Polybrene (Millipore Corporation, Billerica, MA) were used for the infection of RGC-5. After 24 h of growth the efficiency of infection determined as described above was ∼30%. Because pSUPER plasmid contained green fluorescent protein encoding nucleotide sequence, we used flow cytometry (BD-FACSAria Cell Sorting System, BD Biosciences) to enrich the population of cell containing siRNA. |
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