Journal: Frontiers in Physiology

Article Title: Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines

doi: 10.3389/fphys.2023.1221310

Figure Lengend Snippet: iCre/Lox toolkit components available at Addgene.

Article Snippet: To further validate our material and iCre’s efficiency in transgenic experiments, we recombined 469-pME-iCre with p5E-UBI (5′clone containing the Ubiquitin promoter) into the optimized destination clone 1456-pDEST-miniTol2_R4R2_Cryst-eGFP (Addgene # 171795), and injected this final construct 114-UBI:iCre_crystGFP (Addgene #171797) with tol2 transposase to promote genomic integration ( ; ).

Techniques: Sequencing, In Vivo, Expressing, Plasmid Preparation, Construct, Clone Assay, Cloning, Marker, Control, Ubiquitin Proteomics

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids

doi: 10.1007/978-1-0716-2453-1_32

Figure Lengend Snippet: Summary of plasmids described in this work. For each plasmid, the following categories are indicated: plasmid type (Type), plasmid abbreviation (Abbreviation), plasmid description (Description), plasmid function (Function), vector backbone (Backbone), vector resistance (Resistance), and source, including reference.

Article Snippet: MLRV3:HRE-P53-MAPK/JNK-FOXO-TGFβ , Multiplex luciferase reporter vector , Multiplex luciferase reporter vector , Omega Destination-CMV::ELuc:bGHpA , Spectinomycin , Addgene #178320 (this work).

Techniques: Plasmid Preparation, Control, Luciferase, Binding Assay, Multiplex Assay

Journal: The Journal of Experimental Medicine

Article Title: Strictly co-isogenic C57BL/6J- Prnp −/− mice: A rigorous resource for prion science

doi: 10.1084/jem.20151610

Figure Lengend Snippet: TALEN-based generation of C57BL/6J- Prnp ZH3/ZH3 mice. (A) TALEN-binding sites within Prnp exon E3 and start codon (yellow) of the protein coding sequence. Colors indicate the code for repeat-variable diresidues. The Prnp TALEN pair incorporates second-generation heterodimeric FokI cleavage domains ( FokI ELD and FokI KKR ). (B) Sanger sequencing reads of a Prnp WT and a Prnp ZH3 allele from the founder F 0 mouse. A deletion of 8 bp in the Prnp ZH3 allele (highlighted by a red box on the WT sequence) introduces a T/D residue change, followed by a premature STOP codon (*) after residue 14 within the sequence encoding the PrP C signal peptide. The deletion also eliminates the Tsp45I recognition sequence (blue letters on WT sequence). As a result of sequence characteristics in this region, an alternative 8-bp deletion (ACTATGTG), shifted by 4 bp in respect to the previous deletion, is also compatible with the generation of the Prnp ZH3 allele. (C) Representative image of routinely used RFLP analysis discriminating Prnp WT/WT (digested amplicons), Prnp WT/ZH3 (digested and undigested amplicons), and Prnp ZH3/ZH3 mice (only undigested amplicon). Primers location, restriction site, and expected sizes for digestion products are indicated on top of the gel image. (D) Allelic discrimination genotyping using a FAM-labeled WT-specific probe and a Yakima Yellow–labeled ZH3-specific probe. NTC, no-template control. ΔRn, difference in normalized reporter fluorescence after and before amplification. Apart from NTC, each triangle denotes one mouse ( n = 4 mice/genotype). The mean (triangle) and SD (blue error bars) for four technical replicates of each mouse/NTC sample are shown. (E) Immunoblot analysis of PrP C expression in different CNS regions (Cx, cortex; Sc, spinal cord; Cb, cerebellum) of Prnp WT/WT (WT) and Prnp ZH3/ZH3 (ZH3) mice was performed using POM1 (against helices α1 and α3 of the PrP C globular domain). The blot was also decorated with anti-actin antibody as control. (F) Brain PrP C levels as determined by sandwich POM1-POM2 ELISA. Prnp Edbg/Edbg (Edbg) served as negative controls. Each circle denotes a mouse ( n = 3 mice/genotype). Horizontal bar indicates mean. WT→KO, consecutive log 2 dilutions of Prnp WT/WT into Prnp Edbg/Edbg homogenate, indicating that the threshold of detectability was 1:16. (G) Immunofluorescence staining of cerebelli. MAP2 is displayed in green, PrP C , detected with POM19 (against helices β1 and α3 of globular domain) in red, and DAPI in blue. Bar, 20 µm. (D–G) Representative data from two independent experiments.

Article Snippet: TALENs were assembled using the Golden Gate TALEN and TAL Effector kit (plasmid kit 1000000024; Addgene; ) and the pCAG-T7 heterodimeric TALEN destination vectors (plasmids 40131 and 40132; Addgene; ).

Techniques: Binding Assay, Sequencing, Residue, Amplification, Labeling, Control, Fluorescence, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Strictly co-isogenic C57BL/6J- Prnp −/− mice: A rigorous resource for prion science

doi: 10.1084/jem.20151610

Figure Lengend Snippet: C57BL/6- Prnp ZH3/ZH3 mice do not have TALEN off-target cleavage sites. T7 endonuclease I digestion of PCR products generated from predicted OTs ( Table S1 ) and Prnp as a digestion positive control. Analyses were performed on the founder Prnp WT/ZH3 mouse and one control C57BL/6J mouse. Before enzymatic digestion, amplicons were subjected to a temperature gradient enabling the formation of heteroduplexes in the presence of heterozygous mutations in the amplified gDNA. In the presence of TALEN-induced mutations, fragments of the size indicated below the gels are expected to appear, in addition to the undigested, WT amplicon. Nonconsecutive lanes from the same gel show Prnp amplicon as a control. Only in the founder Prnp WT/ZH3 mouse T7 endonuclease I digestion of the Prnp amplicon results in the formation of the two predicted fragments (indicated by an asterisk).

Article Snippet: TALENs were assembled using the Golden Gate TALEN and TAL Effector kit (plasmid kit 1000000024; Addgene; ) and the pCAG-T7 heterodimeric TALEN destination vectors (plasmids 40131 and 40132; Addgene; ).

Techniques: Generated, Positive Control, Control, Amplification