addgene plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: Post-Synaptic Density Proteins in Oligodendrocytes are Required for Activity-Dependent Myelin Sheath Growth

doi: 10.1101/2025.02.10.637467

Figure Lengend Snippet: A. Representation of the plasmid micro-injected into one-cell stage of Tg(s1020t:Gal4) (B, C), Tg(Olig1:KalTA4) (D, E) or Tg(Mbp:Gal4) (F-I) to induce cell-specific dlg4a loss-of-function with the Cas9 endonuclease. B-C. Quantitative RT-PCR, on 1020+ mCherry+ whole larvae, of (B) dlg4a and (C) dlg4b gene expression at 5dpf. Student’s t test; N=4 versus 7 groups of animals, each group containing RNA from 20 dissociated fish. Data are represented as mean ± SD. D. Olig1+ cells in control and dlg4 KO spinal cords at 5 dpf exhibit distinct morphologies. In dlg4a KO, Olig1+ cells are more ramified than in the control. Mature myelinating OLs were mainly observed in control animals. E. Quantification of the oligodendroglial cell surface in control and dlg4a KO animals with IMARIS. Mann-Whitney test; N=117 vs 162 cells, respectively in control and KO fish. F. MBP+ cells with myelin segments in control and KO spinal cords at 5 dpf. G. Quantification of the number of myelin segments / cell. Mann-Whitney test; N=20 vs 30 cells, respectively. H. Quantification the internode length. Mann-Whitney test; N=107 vs 165 internodes. I. Quantification of the internode length per cell. Mann-Whitney test; N=36 vs 41 cells.

Article Snippet: To generate UAS:Cas9-P2A-mCherry; U6-dlg4; U6-dlg4 tol2-flanked cell-specific CRISPR-Cas9 plasmid, we started from the commercially available plasmid pUAS:Cas9-T2A-GFP ; U6-sgRNA1; U6-sgRNA2 (Cat #74009, Addgene) generated by Dr F. Del Bene .

Techniques: Plasmid Preparation, Injection, Quantitative RT-PCR, Expressing, Control, MANN-WHITNEY

Journal: Biology Open

Article Title: EFEMP1 contributes to light-dependent ocular growth in zebrafish

doi: 10.1242/bio.061741

Figure Lengend Snippet: Genotypic details of retina-specific efemp1 -modified mutants ( efemp1 2C-Cas9 ) and phenotypic verification via optomotor responses at 5 days post-fertilization (dpf). (A) The efemp1 2C-Cas9 fish were generated using 2C-Cas9 somatic gene editing. Mutant fish have three separate transgenes: Tg( rx2:Gal4 )×Tg( bact2-loxP-mCherry-loxP-eGFP )×Tg( UAS:Cas9T2ACre;U6:efemp1sgRNA1;U6:efemp1sgRNA2 ). Co-expression of these transgenic elements result in expression of green fluorescence (eGFP) in zebrafish retina, indicative of successful Cas9 protein expression. (A′) Representative retinal image from a 6 dpf fish. Nuclei stained with DAPI are shown in grey. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 40 μm. (B) The universally expressed efemp1 sgRNA1 and sgRNA2 from the transgene binds with Cas9 nucleases and cuts exons 3 and 5 of the efemp1 genome DNA (gDNA), respectively. Standard PCR primers used for genotyping the target sites 1 and 2 amplified 525- (Product 1, P1; spinning from the introns before to after the exon 3) and 471-base pairs (bp; P2; spinning from the intron before to the end of exon 5) of DNA sequences, respectively. Products of headloop PCR are 23-bp longer for P1 and 20-bp longer for P2 due to the headloop tags in reverse primers. UTR, untranslated region. (C,D) Gel electrophoresis of the products from standard and headloop PCR for genotyping targeted sites 1 (C) and 2 (D). In both images, the middle lanes show a 100-bp reference ladder; positions of 300 and 500 bp of size are indicated for both gel images. (E–H) Results of optomotor responses. Spatial-frequency tuning functions (E) for 5 dpf efemp1 +/+ ( n =14) and efemp1 2C-Cas9 fish ( n =13) are three-parameter log-Gaussian functions fit to the data by minimizing the least-square error. The fitted parameters, including (F) normalized amplitude, (G) peak frequency and (H) bandwidth, were compared between groups. Group data are shown as mean±s.e.m. in E and mean with 95% confidence intervals in F–H. * P <0.05 ( F -test).

Article Snippet: The p UAS : Cas9T2ACre ; U6 : sgRNA1 ; U6 : sgRNA2 plasmid (Addgene, #74010) was digested using BsaI (R0535S; NEB, Ipswich, MA, USA) and BsmBI-v2 enzymes (R0739S; NEB) targeting the restriction sites behind the U6 promoters to remove the pre-set sequences that are to be replaced by DNA sequences of sgRNAs.

Techniques: Modification, Generated, Mutagenesis, Expressing, Transgenic Assay, Fluorescence, Staining, Amplification, Nucleic Acid Electrophoresis