econi  (New England Biolabs)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Benefits of therapy by dynamin-2-mutant-specific silencing are maintained with time in a mouse model of dominant centronuclear myopathy

doi: 10.1016/j.omtn.2022.02.009

Figure Lengend Snippet: Specific silencing on mutant allele and protein is maintained over time (A) Semi-quantitative Dnm2 and Atpase RT-PCR products and quantification of Dnm2 expression normalized to Atpase (HTZ n ≥ 10; WT n = 12). (B) EcoNI digestion profile of Dnm2 PCR products and quantification of the mutant/WT, mutant/ Atpase , and WT/ Atpase ratios (HTZ and WT n = 11). (C) DNM2 western blot and quantification of signal by densitometry. GAPDH was used as loading control (HTZ and WT n = 8). In scatterplots (A–C), the bars are mean values and error bars indicate SEM. ∗∗∗p

Article Snippet: For this assay, half of the PCR products were digested using 2U of EcoNI (New England Biolabs, France) for 2 h at 37°C.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Benefits of therapy by dynamin-2-mutant-specific silencing are maintained with time in a mouse model of dominant centronuclear myopathy

doi: 10.1016/j.omtn.2022.02.009

Figure Lengend Snippet: High dose of therapeutic AAV-sh9 does not improve the benefit in old mice (A) Muscle mass relative to body weight in milligrams and grams, absolute maximal force in grams, and specific maximal force (in grams/mg) developed by TAs from WT and AAV-injected HTZ mice (WT n = 8, HTZ-control n = 7, and HTZ-sh9 n = 4). (B) Quantification of Dnm2 expression normalized to Atpase from semi-quantitative Dnm2 and Atpase RT-PCR (left) and quantification of the mutant/WT ratio from EcoNI digestion profile of Dnm2 PCR (right; WT ≥ 4, HTZ-Ctl n = 4, and HTZ-sh9 = 4). (C) Quantification of the vgs per nanogram of DNA in HTZ 6-month-old mice injected with AAV1-sh9 at low (n = 4) or high dose (n = 4). (D) Representative H E staining of TA sections from WT and AAV-injected HTZ mice. Scale bars represent 50 μm. In histogram and scatterplots (A–C), bars represent mean ± SEM. Statistical analysis was performed using Mann-Whitney U test. ∗p

Article Snippet: For this assay, half of the PCR products were digested using 2U of EcoNI (New England Biolabs, France) for 2 h at 37°C.

Techniques: Mouse Assay, Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Polymerase Chain Reaction, Staining, MANN-WHITNEY

Journal: Analytical Chemistry

Article Title: Sequence-Specific DNA Detection at 10 fM by Electromechanical Signal Transduction

doi: 10.1021/ac5021408

Figure Lengend Snippet: Schematic of DNA oligomer preparation. (a) Purified pET-21b plasmids were enzymatically digested by selected pairs of ScaI, PvuI, Pst I, BsaI, and EcoNI restriction enzymes, producing fragments of different lengths. The target DNA sequence complementary to the PNA probe is located beginning at plasmid position 4427 (orange band). Plasmid digestion by ScaI and PvuI produced a 110-base, target-containing fragment, T1. Plasmid digestion by PvuI and PstI produced a 125-base, target-free control fragment, C1. Other fragments were produced similarly: T2 (235 bases) using ScaI and PstI, T3 (419 bases) using ScaI and BsaI, T4 (1613 bases) using by PvuI and EcoNI), C2 (184 bases) using PstI and BsaI, C3 (309 bases) using PvuI and BsaI, and C4 (1503 bases) using ScaI and EcoNI. (b) Following digestion, the DNA was isolated by gel electrophoresis, extracted, and purified. (c) Purified double-stranded DNA was denatured and hybridized with bead–PNA probe conjugates. (d) DNA–PNA–bead mixture was injected into the micropipette for electrical detection.

Article Snippet: Restriction endonucleases ScaI, PvuI, PstI, BsaI, and EcoNI were obtained from New England BioLabs, Inc. (Ipswich, MA).

Techniques: Purification, Positron Emission Tomography, Sequencing, Plasmid Preparation, Produced, Isolation, Nucleic Acid Electrophoresis, Injection

Journal: EMBO Molecular Medicine

Article Title: Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy

doi: 10.15252/emmm.201707988

Figure Lengend Snippet: Si9 and si10 are potent allele‐specific si RNA s in MEF s Semi‐quantitative Dnm2 and Gapdh RT–PCR products and quantification of Dnm2 expression normalized to Gapdh . Sequence of Dnm2 amplicons from cells transfected with si9, si10 and scramble siRNAs. Squares indicate the mutant nucleotide (N = T and A). EcoNI digestion profile Dnm2 PCR and quantification of the mutant/WT ratio. Dnm2 Western blot and quantification of signal by densitometry. Gapdh was used as loading control. Data information: In scatter plots (A, C and D), the bars are mean values and error bars indicate SEM. ** P

Article Snippet: For these assays, half of the PCR products was digested using 2 U of EcoNI (New England Biolabs, France) or PfoI (ThermoFisher Scientific, France) for 2 h at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing, Transfection, Mutagenesis, Polymerase Chain Reaction, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy

doi: 10.15252/emmm.201707988

Figure Lengend Snippet: Sh9 is less efficient in old mice Dnm2 and Gapdh RT–PCR products from TA muscles and quantification of Dnm2 expression normalized to Gapdh . WT muscles were included as control. EcoNI digestion profile of the Dnm2 amplicons and quantification of the mutant/WT ratio ( n = 6). Histochemical staining of TA sections from HTZ mice injected with AAV‐sh9 or AAV‐control. DPNH: reduced diphosphopyridine nucleotide diaphorase staining. Asterisks indicate fibres with abnormal central accumulations. Scale bars = 50 μm. Quantification of histological abnormality. Scatter plot represents individual percentages of histological abnormality from heterozygous control or treated mice ( n = 6). Muscle mass normalized by the total body weight (mg/g) in AAV‐injected mice ( n = 6 for WT and n = 12 for HTZ). Absolute maximal force (F) and specific maximal force (G) developed by TA muscles ( n = 6 for WT and n ≥ 11 for HTZ). Data information: In (A, B, D–G), the bars in scatter plots represent the mean ± SEM. Statistical analysis was performed using a one‐tailed Mann–Whitney U ‐test. * P

Article Snippet: For these assays, half of the PCR products was digested using 2 U of EcoNI (New England Biolabs, France) or PfoI (ThermoFisher Scientific, France) for 2 h at 37°C.

Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis, Staining, Injection, One-tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy

doi: 10.15252/emmm.201707988

Figure Lengend Snippet: Sh9 is efficient to specifically reduce the mutant allele in 3‐month‐treated muscle from young mice Dnm2 and Gapdh RT–PCR products from AAV‐shRNA‐transduced muscles and quantification of Dnm2 expression normalized to Gapdh . WT muscles were included as control. Sequence of Dnm2 amplicons from transduced TA muscles. Squares indicate the mutant nucleotide (N = T and A). EcoNI digestion profile of the Dnm2 amplicons and quantification of the mutant/WT ratio. Data information: In scatter plots (A, C), the bars are mean values and error bars indicate SEM. * P

Article Snippet: For these assays, half of the PCR products was digested using 2 U of EcoNI (New England Biolabs, France) or PfoI (ThermoFisher Scientific, France) for 2 h at 37°C.

Techniques: Mutagenesis, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, shRNA, Expressing, Sequencing

Journal: EMBO Molecular Medicine

Article Title: Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy

doi: 10.15252/emmm.201707988

Figure Lengend Snippet: Identification of six allele‐specific si RNA s in MEF s Wild‐type (WT) and mutant (R465W) mRNA sequences in the region of the mutation. The sequences of the 19 possible siRNAs targeting the mutation (in red) are indicated. Arrows show the sense strand of the 12 assessed siRNAs numbered relative to the position of the mismatch between siRNA and WT sequences. EcoNI digestion profile of the Dnm2 RT–PCR products. Histogram represents mean ± SEM of calculated mutant/WT ratio for siRNAs transfected at 20 nM for 48 h. * P

Article Snippet: For these assays, half of the PCR products was digested using 2 U of EcoNI (New England Biolabs, France) or PfoI (ThermoFisher Scientific, France) for 2 h at 37°C.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Transfection