Journal: Journal of Veterinary Science

Article Title: Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

doi: 10.4142/jvs.2016.17.1.89

Figure Lengend Snippet: Generation of porcine α1,3-galactosyltransferase ( GGTA1 ) knockout (KO) fibroblasts with transcription activator-like effector nucleases (TALENs). (A) Sequences of the TALEN binding site in the GGTA1 gene. (B) TALEN driven GGTA1 mutations detected by the T7 endonuclease I (T7E1) assay in a cell population isolated using a biotin-labeled IB4 lectin attached to dynabeads magnetic beads. (C) DNA sequencing of TALEN target region in transfected cells. WT, wild type.

Article Snippet: Briefly, the purified PCR products from the DNA isolated from colonies were denatured at 95°C for 5 min, then re-annealed at room temperature for 10 min, after which they were digested by T7E1 (ToolGen, Korea) at 37°C for 30 min. Digestion of the PCR product was expected if the colony contained mutated GGTA1 .

Techniques: Knock-Out, TALENs, Binding Assay, Isolation, Labeling, Magnetic Beads, DNA Sequencing, Transfection

Journal: Nucleic Acids Research

Article Title: qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites

doi: 10.1093/nar/gky505

Figure Lengend Snippet: Analysis of the Cas9-induced target modification efficiency in GFP-positive cells. The GFP signal indicates cells carrying the Cas9_E4.2 plasmid transfected either by electroporation (left-hand side) or lipofection (right-hand side). Based on the fluorescence intensity, GFP-positive cells were divided into three arbitrarily designated categories: low, medium, and high. ( A ) The TME bar plots depicting the results of qEva-CRISPR analysis of low, medium, and high fractions of the HCT116 GFP-positive cells. The chart designations are as shown in Figure . ( B ) Agarose gels depicting the results of T7 endonuclease I (T7E1) analysis of DNA samples from the above-described cells. Bands representing full-length (410 bp) and digested PCR products (∼267 bp and 143 bp) are indicated on the left. Only heteroduplexes with Cas9-induced mismatches are digested by T7E1. PCR products digested (+) and not digested (–) with T7E1 are shown next to each other. Control – PCR product of DNA samples extracted from cells not transfected with Cas9_E4.2 plasmid; L – 1 Kb Plus DNA ladder (ThermoFisher Scientific, Waltham, MA). The percentage of digested products (calculated based on the densitometric analysis) corresponding to a fraction of the Cas9-mutated targets are shown in the particular lines on agarose gels (red fonts).

Article Snippet: The most popular of these techniques utilize T7 endonuclease 1 (T7E1) or Surveyor nuclease (Transgenomic, Inc., USA) to cleave mismatches formed between modified and unmodified DNA strands ( , ).

Techniques: Modification, Plasmid Preparation, Transfection, Electroporation, Fluorescence, CRISPR

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: CRISPR/Cas9-based liver-derived reporter cells for screening of mPGES-1 inhibitors

doi: 10.1080/14756366.2019.1587416

Figure Lengend Snippet: List of oligonucleotides used in the present study. ^a

Article Snippet: Cellular DNA was extracted 48 h after co-transfection of PX459:sgRNA2 and donor according to the T7E1 test kit instructions (GenePharma, Shanghai, China), using the T7E1 sgRNA targeting test F′ and R′ primers ( ).

Techniques: Sequencing, Plasmid Preparation, Quantitative RT-PCR

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: CRISPR/Cas9-based liver-derived reporter cells for screening of mPGES-1 inhibitors

doi: 10.1080/14756366.2019.1587416

Figure Lengend Snippet: Construction of mPGES-1 fluorescent reporter cells using CRISPR/Cas9 technology. (A) CRISPR/Cas9 knock-in was used to construct mPGES-1 fluorescent reporter cells. 2A-tdTomato-loxp-CAG-Neo-loxp was integrated into the PTGES gene of chromosome to replace the stop codon to obtain the reporter cells stably expressing red fluorescence and G418 resistance. (B) Six sgRNAs were distributed in different positions of PTGES gene. (C) PX459: sgRNAs were transiently transfected into 293T cells, and DNA and RNA were extracted 48 h later. Three micrograms of RNA was reverse transcribed for real-time fluorescent quantitative PCR, and the group transfected with PX459 empty vector was used as a control. The value was set to 1, and * p < .05, ** p < .005, n = 3. The extracted DNA was used for T7E1 assay, the red arrow was the strip after digestion, – was the negative control, and + was the positive control. (D) The basic expression of mPGES-1 protein in different hepatogenic cells. Thirty micrograms of total protein was used for Western blot, and β-actin was used as internal reference protein to normalise gray value. (E) The construction process of monoclonal fluorescent reporter cells. PX459: sgRNA2 and donor vector were co-transfected into cells, and screened by G418 for 2 weeks. Cells were picked in 96-well plates and expanded to obtain monoclonal cells stably expressing red fluorescence for subsequent validation. (F) Cells stably expressing red fluorescent protein were obtained by G418 screening.

Article Snippet: Cellular DNA was extracted 48 h after co-transfection of PX459:sgRNA2 and donor according to the T7E1 test kit instructions (GenePharma, Shanghai, China), using the T7E1 sgRNA targeting test F′ and R′ primers ( ).

Techniques: CRISPR, Knock-In, Construct, Stable Transfection, Expressing, Fluorescence, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Stripping Membranes, Negative Control, Positive Control, Western Blot, Biomarker Discovery