Journal: Brain

Article Title: Stepwise crosstalk between aberrant Nf1, Tp53 and Rb signalling pathways induces gliomagenesis in zebrafish

doi: 10.1093/brain/awaa404

Figure Lengend Snippet: CRISPR/Cas9-based deletion system in zebrafish. ( A ) Schematic illustration of the constructs of the CRISPR/Cas9-based vector, including the gfap promoter-driving Cas9, mCherry reporter, and U6-driving specific gRNA. ( B ) Strategy of the generation of the transgenic zebrafish lines. ( C ) F 1 transgenic fish generated by mosaic mCherry-positive fish outcrossed with wild-type fish. A subset of the transgenic offspring was identified by mCherry-positive astrocytes. Scale bar = 1 mm. ( D ) The colocalization of mCherry-labelled Cas9 and GFP-labelled endogenous Gfap in 60 hpf offspring from the cross of gfap WT and Tg ( gfap : GFP ) transgenic fish. Scale bars = 500 μm. ( E – J ) Co-localization of mCherry fluorescent signals (red) and endogenous Gfap detected by anti-Gfap antibody (FITC; green) in the brain and retina ( E – G ) or spinal cord ( H – J ) tissue. Scale bars = 200 μm. ( K ) Representative images of whole-mount in situ hybridization using an anti-sense RNA probe against Cas9 mRNA in 48 hpf embryos injected with Tol2 mRNA and gfap : Cas9-T2A-mCherry,U6 : gRNA ( null ) vector expressing Cas9 under the control of the gfap promoter. Cas9 expression pattern is governed by the tissue-specificity of gfap promoter (yellow arrowheads). Scale bars = 500 μm.

Article Snippet: We cloned the zebrafish U6-3 promoter ( Halbig et al. , 2008 ) from the AB strain, followed by an NheI site, into the CRISPR/Cas9 skeleton vector ( Cas9-T2A-mCherry ) from the Tol2 kit ( Kwan et al ., 2007 ) using ClaI and KpnI endonucleases (New England Biolabs).

Techniques: CRISPR, Construct, Plasmid Preparation, Transgenic Assay, Generated, In Situ Hybridization, Injection, Expressing

Journal: Bioactive Materials

Article Title: 3D bioprinting of a biomimetic meniscal scaffold for application in tissue engineering

doi: 10.1016/j.bioactmat.2020.11.027

Figure Lengend Snippet: Cytocompatibility of the meniscus-derived bioink. (a) CCK-8 assays results of MFCs cocultured with different hydrogels for 1–7 days. (b) Comparative gene expression analysis for chondrogenic SOX9, COL1A2 and COL2A1 in GelMA and GelMA/MECM at 14days. (c) Immunofluorescence images showing the chondrogenic phenotype of MFCs in GelMA and GelMA/MECM constructs by COL type I staining (Green), cell nuclei (DAPI, blue) and F-actin (Rhodamine-phalloidin, red) (scale bar: 500 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The samples were cultured for the specific time, and then fixation, permeabilization and blocking were performed followed by incubation with primary antibodies (1:500) (Novus, USA) and rhodamine-phalloidin (1:200) (Cytoskeleton, USA) for overnight at 37 °C, and with secondary antibodies (1:200) (ZSGB-BIO, China) for 1 h at room temperature.

Techniques: Derivative Assay, CCK-8 Assay, Expressing, Immunofluorescence, Construct, Staining