oligonucleotide dna sequences encoding shrnas against htt  (Thermo Fisher)

Journal:

Article Title: 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

doi: 10.1016/j.bbrc.2010.05.092

Figure Lengend Snippet: A. The genomic structure of the human CDK9 gene and transcription start sites of mRNAs encoding the 55K and 42K proteins are shown (24). The sequence of the wild type 55K mRNA and the sequence of the mRNA encoding the shRNA-resistant 55K protein, termed 55Kr, are shown. B. At four days post-infection, HeLa cell extracts were prepared from cultures infected in duplicate with 55K-shRNA lentivirus, parental Vector-shRNA lentivirus; a single culture was mock-infected. Expression levels of the indicated proteins were examined in immunoblots. C. Expression of the eGFP reporter protein in the cultures infected with the indicated shRNA viruses was examined by flow cytometry at two, four, and seven days post-infection (top panel). Portions of cell cultures were also stained with propidium iodide; the percentage of cells that had a sub-2N DNA content as determined by flow cytometry are shown (bottom panel).

Article Snippet: DNA oligonucleotides encoding shRNA sequences were purchased from Invitrogen, and inserted under the control of the U6 promoter in the FG12 lentiviral vector ( 12 ).

Techniques: Sequencing, shRNA, Infection, Plasmid Preparation, Expressing, Western Blot, Flow Cytometry, Staining

Journal:

Article Title: 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

doi: 10.1016/j.bbrc.2010.05.092

Figure Lengend Snippet: A. HeLa cells were infected with the 55K-shRNA or Vector-shRNA lentiviruses and at two days post-transduction were processed for indirect immunofluorescence to detect γ-H2AX to monitor DSBs and stained with DAPI to visualize DNA. B. Pools of puromycin-resistant HeLa cells over-expressing the wild type 55K CDK9 (puro55K-WT) or the shRNA-resistant CDK9 protein (Puro55Kr) were infected with the 55K-shRNA, Con-shRNA, or parental lentiviral vector At day four post-infection cell extracts were prepared and the indicated proteins evaluated in immunoblots.

Article Snippet: DNA oligonucleotides encoding shRNA sequences were purchased from Invitrogen, and inserted under the control of the U6 promoter in the FG12 lentiviral vector ( 12 ).

Techniques: Infection, shRNA, Plasmid Preparation, Transduction, Immunofluorescence, Staining, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: L1 retrotransposon-mediated stable gene silencing

doi: 10.1093/nar/gni056

Figure Lengend Snippet: L1 retrotransposon-based RNAi system. ( A ) Mechanism of L1 retrotransposition. ( B ) Strategy for L1-based RNAi. A selectable neo -cassette for retrotransposition is inserted in the 3′-UTR of L1 RP , followed by an RNAi-cassette composed of the H1 promoter, the 19 bp sense and antisense sequences of a specific gene separated by a 9 bp loop sequence and a pol III terminator of six thymidines. Abbreviations: pA, polyadenylation signal; NEO, neo gene; SD, splicing donor site; and SA, splicing acceptor site. Arrows indicate transcription start sites. ( C ) Structure of L1-based RNAi vector (pL1-Silencer) and expressed shRNA. ( D ) L1-based RNAi system. Transfection of pL1-Silencer into cultured cells is followed by the transcription of L1-RNAi sequence, its integration into the host genome and stable siRNA expression.

Article Snippet: DNA oligonucleotides encoding control shRNA sequence, 5′-ACTACCGTTGTTATAGGTG-3′, which expresses an siRNA with limited homology to any known sequences in the human, mouse and rat genomes (Ambion, Austin, TX), green fluorescence protein (GFP) target sequence, 5′-GGCTACGTCCAGGAGCGCA-3′ , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) target sequence, 5′-GTGGATATTGTTGCCATCA-3′ (Ambion), were synthesized by Integrated DNA Technologies (Coralville, IA).

Techniques: Sequencing, Plasmid Preparation, shRNA, Transfection, Cell Culture, Expressing

Journal: Nucleic Acids Research

Article Title: L1 retrotransposon-mediated stable gene silencing

doi: 10.1093/nar/gni056

Figure Lengend Snippet: Inhibition of exogenous GFP expression using L1-based RNAi system. ( A ) Real-time RT–PCR results of GFP mRNA expression in stable HeLa lines expressing control or GFP-targeted siRNA ( n = 6). ( B ) Western-blot analysis of GFP expression in control or knockdown clones. ( C ) Typical FACS dot plots of GFP expression in control and knockdown clones. ( D ) Normalized GFP fluorescence in control and knockdown clones by FACS ( n = 4).

Article Snippet: DNA oligonucleotides encoding control shRNA sequence, 5′-ACTACCGTTGTTATAGGTG-3′, which expresses an siRNA with limited homology to any known sequences in the human, mouse and rat genomes (Ambion, Austin, TX), green fluorescence protein (GFP) target sequence, 5′-GGCTACGTCCAGGAGCGCA-3′ , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) target sequence, 5′-GTGGATATTGTTGCCATCA-3′ (Ambion), were synthesized by Integrated DNA Technologies (Coralville, IA).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Control, Western Blot, Knockdown, Clone Assay, Fluorescence

Journal: Nucleic Acids Research

Article Title: L1 retrotransposon-mediated stable gene silencing

doi: 10.1093/nar/gni056

Figure Lengend Snippet: Inhibition of endogenous GAPDH expression using L1-based RNAi system. ( A ) Real-time RT–PCR results of GAPDH mRNA expression in stable HeLa lines expressing control, GFP-targeted or GAPDH-targeted siRNA ( n = 6). ( B ) Western-blot analysis of GAPDH protein expression in control or GAPDH knockdown clones. ( C ) Immunofluorescent staining of GAPDH (green) and cytokeratin (red) in control and knockdown cells. Nucleus is counterstained with DAPI (blue).

Article Snippet: DNA oligonucleotides encoding control shRNA sequence, 5′-ACTACCGTTGTTATAGGTG-3′, which expresses an siRNA with limited homology to any known sequences in the human, mouse and rat genomes (Ambion, Austin, TX), green fluorescence protein (GFP) target sequence, 5′-GGCTACGTCCAGGAGCGCA-3′ , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) target sequence, 5′-GTGGATATTGTTGCCATCA-3′ (Ambion), were synthesized by Integrated DNA Technologies (Coralville, IA).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Control, Western Blot, Knockdown, Clone Assay, Staining

Journal: Nucleic Acids Research

Article Title: L1 retrotransposon-mediated stable gene silencing

doi: 10.1093/nar/gni056

Figure Lengend Snippet: Northern-blot analysis of siRNA expression in GAPDH knockdown cells. Upper panel (from left to right): lane 1, 19 nt probe containing the sense strand sequence of GAPDH siRNA as a molecular weight marker (MW), which was also used as the probe to detect antisense siRNA in the test samples; lane 2: wild-type HeLa (wt); lanes 3–6: four GAPDH knockdown lines (#1, 2, 3 and 4). Position of antisense siRNA is indicated. Note the inverse relationship of siRNA expression seen here with GAPDH mRNA and protein expression . Lower panel: gel post-electrophoresis. Positions of 5.8S, 5S and tRNAs are indicated.

Article Snippet: DNA oligonucleotides encoding control shRNA sequence, 5′-ACTACCGTTGTTATAGGTG-3′, which expresses an siRNA with limited homology to any known sequences in the human, mouse and rat genomes (Ambion, Austin, TX), green fluorescence protein (GFP) target sequence, 5′-GGCTACGTCCAGGAGCGCA-3′ , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) target sequence, 5′-GTGGATATTGTTGCCATCA-3′ (Ambion), were synthesized by Integrated DNA Technologies (Coralville, IA).

Techniques: Northern Blot, Expressing, Knockdown, Sequencing, Molecular Weight, Marker, Electrophoresis