pgl3 cloning vector  (Boehringer Mannheim)

Journal: Cells

Article Title: Dietary Choline Mitigates High-Fat Diet-Impaired Chylomicrons Assembly via UPRer Modulated by perk DNA Methylation

doi: 10.3390/cells11233848

Figure Lengend Snippet: Choline down-regulated perk expression by controlling − 211 site-specific DNA methylation. ( A ) mRNA levels of the genes related to DNA methyltransferase in the intestine. ( B ) Western blot for Dnmt1. ( C ) The histogram of relative methylation level of perk . ( D ) Heat map of relative methylation level of perk . ( E ) The histogram of quantitative methylation percentage of perk in the IECs under FA and CH incubation and transfected with 5-azacitidine (AZA). ( F ) The relative luciferase activities of perk. ( G ) mRNA levels of perk. ( H ) Western blot and quantification analysis for Perk. ( I ) The quantitative methylation percentage of perk − 211 methylation site. ( J ) Site mutation analysis of perk − 211 methylation site on pGl3- perk − 822/+122 vectors. ( K , L ) Density gradient curve and quantification analysis for CMs-TGs/or apolipoprotein in the IECs under FA and CH incubation and transfected with − 211 methylation site mutation. Data are mean ± SEM, n = 3 independent biological experiments; different lower-case letters indicate significant differences in −AZA groups; different capital letters indicate significant differences in +AZA groups ( p ≤ 0.05); asterisks indicate significant differences between −AZA and +AZA groups (* p ≤ 0.05).

Article Snippet: Next, we created perk promoters using the ClonEx-pressTM II One Step Cloning Kit and inserted them into the pGl3 basic vector (C112, Vazyme, Piscataway, NJ, USA).

Techniques: Expressing, DNA Methylation Assay, Western Blot, Methylation, Incubation, Transfection, Luciferase, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic Microinjection

doi: 10.7150/ijbs.35930

Figure Lengend Snippet: Generation of gene-modified Tibet minipigs by Cas9 mRNA/sgRNAs cytoplasmic microinjection. A. Schematic diagram of sgRNAs targeting TYR , IL2RG , and RAG1 loci. The TYR , IL2RG or RAG1 target site(s) were located in exons 1, 3 and 2, respectively. The sgRNA targeting sites are underlined and highlighted in red. Protospacer adjacent motifs (PAM) are indicated in blue. B-C. Schematic diagram of Cas9 mRNA plus sgRNA microinjection into the cytoplasm of 1-cell stage ( B ) or 2-cell stage ( C ) embryos. D. Surrogate A gave birth to four piglets (i.e., a1, a2, a3 and a4). E. Surrogate B gave birth to four piglets (i.e., b5, b6, b7 and b8). F. Surrogate C gave birth to four piglets (i.e., c9, c10, c11 and c12). G. Surrogate D gave birth to four piglets (i.e., d13, d14, d15, and d16).

Article Snippet: The sgRNA cloning vector was pGL3-U6-gRNA-PGK-puromycin (Addgene plasmid 51133), and the Cas9 expressing plasmid was pST1374-NLS-flag-linker-Cas9 (Addgene plasmid 44758).

Techniques: Modification, Microinjection