guide rna sequence targeting 5  (Addgene inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Revealing the biological features of the axolotl pancreas as a new research model

doi: 10.3389/fcell.2025.1531903

Figure Lengend Snippet: The mutations of Pancreatic and duodenal homeobox 1 ( Pdx1 ) result in varying degrees of absence in both the endocrine and exocrine cells of the axolotl pancreas, attributable to different allele mutations. (A) Schematic diagrams of the Pdx1 gene structure and PDX1 protein domains, along with a schematic illustration of the actual effects of Pdx1 mutations on the proteins. CRISPR gRNAs was designed to target the Pdx1 sequence and injected into one-cell-stage eggs, and resulted in mild and severe phenotypes. Axolotls with a complete homeobox in both Pdx1 alleles ( Pdx1 s/s ) exhibit a severe phenotype. In contrast, a mismatch or frameshift mutation in the homeobox region of both Pdx1 alleles ( Pdx1 m/m ) results in a milder pancreatic defect. (B) Fasting blood glucose levels in control, heterozygous, and homozygous axolotls show that blood glucose significantly increase in Pdx1 −/− mutants, while no significant change is observed in Pdx1 +/− mutants. (C–E) Anatomical analysis of control (C) and Pdx1 mutant axolotls (blue circle). A complete pancreatic structure, but with a small portion of the pancreas body missing (D) . While a severe phenotype is characterized by the development of only a remnant of pancreatic head (E) . (F) In situ hybridization for Amy (blue) on cryosections shows that Pdx1 mutants causes a deficiency in the exocrine pancreas. (G) Immunofluorescence for INS (red), GCG (green), combined with DAPI (blue) on cryosections shows INS + /GCG + double hormone cells (arrows) appear in Pdx1 m/m mutant axolotls, while Pdx1 s/s mutant axolotls have no endocrine cells. (H) Statistical analysis of endocrine cell counts shows a decrease in the number of β, α, and δ cells following Pdx1 mutations. (I) Statistical analysis of the proportions of β, α, and δ cells in the control, Pdx1 m/m , and Pdx1 s/s groups. (J) Statistical analysis of double hormone cells in the control and Pdx1 m/m groups. (K) Statistical analysis shows that animal growth is severely inhibited in Pdx1 s/s mutant axolotls. Data are the mean ± s. e.m. Scale bar: (C–E) 200 μm; (F) 200 μm; (G) 50 μm.

Article Snippet: The Pdx1 guide RNA (gRNA) sequence (5′-AAG GAG GAG GAC AAG AAG CG-3′) was synthesized by GenScript.

Techniques: CRISPR, Sequencing, Injection, Mutagenesis, Control, In Situ Hybridization, Immunofluorescence

Journal: Frontiers in Microbiology

Article Title: Faecalibacterium prausnitzii promotes intestinal epithelial IL-18 production through activation of the HIF1α pathway

doi: 10.3389/fmicb.2023.1298304

Figure Lengend Snippet: Stable knockout of HIF1A in Caco-2 cells leads to decreased HIF1α signaling upon pathway activation. (A) Time-dependent HIF1α responsive element (HRE)-luciferase assay over 48 h treatment with 1 mM DMOG. Gene expression levels of (B) HIF1A and the HIF1α pathway targets (C) PGK1 , (D) EGLN3 , and (E) IL18 in Caco-2- HIF1A -null and control cells treated with 1 or 5 mM DMOG for 24 h. Data presented as mean ± SD. All experiments were performed with n = 3; * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Single guide RNAs for HIF1A (sequence 5′-GATGGTAAGCCTCATCACAG-3′) were designed via Benchling © online platform ( https://www.benchling.com/ ) and cloned in pLenti-sgRNA backbone (Addgene, 71409).

Techniques: Knock-Out, Activation Assay, Luciferase, Expressing

Journal: Frontiers in Microbiology

Article Title: Faecalibacterium prausnitzii promotes intestinal epithelial IL-18 production through activation of the HIF1α pathway

doi: 10.3389/fmicb.2023.1298304

Figure Lengend Snippet: Differential gene expression (DGE) analysis of Caco-2- HIF1A -null upon coculture with F. prausnitzii , compared to Caco-2 empty vector controls. (A) Schematic representation of the HoxBan coculture system, including experiment layout. Caco-2- HIF1A -null cells, or appropriate control (iCas9 empty vector), were cultured in the HoxBan system for 18 h in the absence or presence of F. prausnitzii inoculum (in figure, mono and co, respectively). Volcano plots showing differential gene expression analysis comparing Caco-2- HIF1A -null cells and Caco-2-empty vector in monoculture ( B ; purple and pink dots represent downregulated and upregulated genes, respectively) and coculture with F. prausnitzii ( C ; green and blue dots represent downregulated and upregulated genes, respectively). All dots display significant observations with p < 0.05. Veen diagrams were used to discover uniquely (D) upregulated and (E) downregulated DGE (numbers underlined inside blue and green diagrams, respectively) in Caco-2- HIF1A -null cultured with F. prausnitzii , compared to Caco-2-empty vector in same condition. All experiments performed in n = 3.

Article Snippet: Single guide RNAs for HIF1A (sequence 5′-GATGGTAAGCCTCATCACAG-3′) were designed via Benchling © online platform ( https://www.benchling.com/ ) and cloned in pLenti-sgRNA backbone (Addgene, 71409).

Techniques: Expressing, Plasmid Preparation, Cell Culture

Journal: Frontiers in Microbiology

Article Title: Faecalibacterium prausnitzii promotes intestinal epithelial IL-18 production through activation of the HIF1α pathway

doi: 10.3389/fmicb.2023.1298304

Figure Lengend Snippet: Uniquely upregulated genes in F. prausnitzii -cocultured Caco-2- HIF1A -null cells, compared to F. prausnitzii -cocultured Caco-2 control cells.

Article Snippet: Single guide RNAs for HIF1A (sequence 5′-GATGGTAAGCCTCATCACAG-3′) were designed via Benchling © online platform ( https://www.benchling.com/ ) and cloned in pLenti-sgRNA backbone (Addgene, 71409).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Faecalibacterium prausnitzii promotes intestinal epithelial IL-18 production through activation of the HIF1α pathway

doi: 10.3389/fmicb.2023.1298304

Figure Lengend Snippet: Uniquely downregulated genes in F. prausnitzii -cocultured Caco-2- HIF1A -null cells, compared to F. prausnitzii -cocultured Caco-2 control cells.

Article Snippet: Single guide RNAs for HIF1A (sequence 5′-GATGGTAAGCCTCATCACAG-3′) were designed via Benchling © online platform ( https://www.benchling.com/ ) and cloned in pLenti-sgRNA backbone (Addgene, 71409).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Faecalibacterium prausnitzii promotes intestinal epithelial IL-18 production through activation of the HIF1α pathway

doi: 10.3389/fmicb.2023.1298304

Figure Lengend Snippet: RNA sequencing analysis reveals that HIF1A ablation prevents upregulation of IL18 by F. prausnitzii in intestinal epithelial cells. (A,B) HIF1α and HIF2α scores shows a decrease in HIF1α, but not HIF2α, activation capacity in Caco-2- HIF1A -null, compared to Caco-2-empty vector control in coculture with F. prausnitzii . (C) Normalized gene expression counts of IL18 in Caco-2- HIF1A -null cells, compared to Caco-2-empty vector in monoculture and coculture with F. prausnitzii . (D) Spearman correlation analysis between IL18 gene expression and HIF1α scores on Caco-2 cells in the HoxBan system. All experiments performed in n = 3 (ns = not significant, ** p < 0.001 and *** p < 0.0001).

Article Snippet: Single guide RNAs for HIF1A (sequence 5′-GATGGTAAGCCTCATCACAG-3′) were designed via Benchling © online platform ( https://www.benchling.com/ ) and cloned in pLenti-sgRNA backbone (Addgene, 71409).

Techniques: RNA Sequencing Assay, Activation Assay, Plasmid Preparation, Expressing