Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: Glycan substrates and relative activity for Endo S and Endo S2.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: Activity Assay

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: In all cases, digestions were performed at 37 °C for 20 min with 0.5 μg of the indicated enzyme in 20 μL buffer of 50 mM MES pH 6.0 and separated on 17% SDS-gel. Both glycan fluorescent images and protein images of the same gels are shown. N2f was run in all three groups and served as a control. a General scheme for Endo S/S2 and Endo-β-Gal digestions. Digestion by Endo S/S2 results in the product Fuc-α,6-GlcNAc and digestion by Endo-β-Gal results in product N2f. b Digestion of Group I glycans by all three enzymes. While Endo S2 showed complete digestion on all substrates, Endo S only showed complete digestion on N2f and G2f. Endo-β-Gal only digested xG2f and xxG2f. c Digestion of Group II glycans by Endo S/S2. Slight product formation was observed when M1N1f was digested by Endo S and M2f was digested by Endo S2. d Digestion of Group III glycans by all three enzymes. No obvious digestion was observed on these substrates. Product formation in N3f digestion by Endo S/S2 was due to the digestion on contaminated N2f that was due to incomplete conversion during synthesis.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: SDS-Gel

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: In each reaction, 2 pmol of a substrate glycan was digested with 500 ng enzyme in 20 μl buffer of indicated pH for 30 min at 37 °C and then separated on 15% gel. Both glycan images and protein images are shown for the assays. a pH profile of Endo S on G2f. b pH profile of Endo S2 on G2f. c pH profile of Endo-β-Gal on xG2f.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques:

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: Different glycans were digested with indicated amounts of Endo S/S2 and separated on 15% gels and imaged for both glycans and proteins. The RA of each measurement are shown. a Relative activity on M1N1f. b Relative activity on N2f. c Relative activity on G2f. d Relative activity on N2nf. Except d where samples were digested for 18 h, all other samples were digested for 1 h at 37 °C.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: Activity Assay

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: a Plot of relative activities of Endo S/S2 on various glycans. Endo S/S2 are most active on N2f and G2f and are much less active on all other glycans. b Endo S/S2 substrate recognition motifs. The impacts of individual monosaccharide modification introduced by various glycosyltransferases on the activity of Endo S/S2 are indicated with arrows, x symbols and folds of increase/decrease. The key residues identified from this study are numbered and possible enzyme recognition motif is highlighted.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: Modification, Activity Assay

Journal: Nature Communications

Article Title: EZH2 loss promotes gastric squamous cell carcinoma

doi: 10.1038/s41467-025-61024-5

Figure Lengend Snippet: a Western blot analysis of EZH2 in sgScr (Cas9-sgScramble), sg Ezh2 -1(Cas9-sg Ezh2 -1), and sg Ezh2 -2(Cas9-sg Ezh2 -2) gastric organoids (−1 and −2 represent two independent sgRNAs targeting Ezh2 ). Representative blot ( a ) of n = 2 technical replicates. b Western blot analysis of H3K27me3 in sgScr, sg Ezh2 -1, and sg Ezh2 -2 gastric organoids. Representative blot ( b ) of n = 2 technical replicates. c Representative bright-field(top), H&E(middle), CK14 and P40 IF staining(bottom) images of sgScr, sg Ezh2 -1, and sg Ezh2 -2 mouse gastric organoids. Scale bars, 50 µm (top) and 20 µm (middle and bottom). d The heatmap showing the differential expression genes ( p < 0.05 and absolute log 2 fold-change > 0.5, p- value was determined by unpaired two-tailed Wald test) between sgScr, sg Ezh2 -1 and sg Ezh2 -2 gastric organoids, measured by RNA-seq analyses. e Statistical graphs showing the diameter of TP ( Trp53 -/- ; sg Pten ) and TPE ( Trp53 -/- ; sg Pten ; sg Ezh2 ) gastric organoids. f Statistical graphs showing the organoids formation rate of TP and TPE gastric organoids ( n = 3 biological replicates). g The bright-field image of subcutaneous tumors of TP ( n = 3 mice) and TPE ( n = 3 mice), Scale bar, 1 cm. h Tumor weight of subcutaneously transplanted TP ( n = 3 mice) and TPE ( n = 3 mice). i H&E staining of subcutaneous tumor tissues of TP and TPE mice. Scale bar, 20 µm. j Representative images of CK14(left), CK5(middle) and P40(right) IF staining of subcutaneous tumor tissues of TP and TPE mice. Scale bars, 20 µm and 50 µm. Data are shown as means ± SD, p- value was determined by unpaired two-tailed t test( e , f , h ). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were H3K27me3 (Abclonal, A2363,1:200), Ezh2 (D2C9) XP® Rabbit mAb (Cell Signaling Technology, 5246S, 1:50 for IHC, 1:1000 for IF), Anti-AP2 gamma/TFAP2C antibody [3B5] (Abcam, ab110635,1:50 for IHC, 1:500 for IF), Cytokeratin 5Monoclonal antibody(2C2) (Invitrogen, MA5-17057, 1:500 for IHC, 1:1000 for IF), Anti-Cytokeratin 14 antibody (Abcam, ab7800, 1:500), Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb (Cell Signaling Technology, 19526S, 1:400), Anti-Cytokeratin 7 antibody [ EPR17078 ] - Cytoskeleton Marker (Abcam, ab181598, 1:4000), CK20 (Abcam, ab53120,1:100), anti-P40-DeltaNp63antibody [EPR17863-47] (Abcam, ab203826, 1:1000 for IHC, 1:50 for IF), Cytokeratin 5/6 Monoclonal antibody (Thermo Fisher Scientific, MA5-12429, 1:200), Anti-MUC1 antibody [EP1024Y] (Abcam, ab45167, 1:400), MUC5AC monoclonal antibody (45M1) (Thermo Fisher Scientific, MA5-12178, 1:400).

Techniques: Western Blot, Staining, Quantitative Proteomics, Two Tailed Test, RNA Sequencing

Journal: Nature Communications

Article Title: EZH2 loss promotes gastric squamous cell carcinoma

doi: 10.1038/s41467-025-61024-5

Figure Lengend Snippet: a Survival curve of mice orthotopically transplanted with TP, TPE-1, and TPE-2 organoids ( n = 6 mice). All curves were analyzed by log-rank (Mentel-Cox) test (− 1 and − 2 represent two independent sgRNAs targeting Ezh2 ). b Representative bright-field (top) and red fluorescence(bottom) images of gastric cancer transduced with TP, TPE-1 and TPE-2 organoids. Scale bar, 1 mm. c Statistical graphs showing the tumor weight of mice orthotopically transplanted with TP, TPE-1 and TPE-2 organoids ( n = 6 mice). d Representative IHC staining of EZH2 in gastric tumor sections of TP (left), TPE-1 (middle), and TPE-2(right) mice. Scale bar, 20 µm. e Western blot analysis of EZH2 in TP, TPE-1 and TPE-2 mouse gastric tumor organoids. Representative blot ( e ) of n = 2 biological replicates. f Representative IHC staining of H3K27me3 in gastric cancer sections of TP (left), TPE-1 (middle), and TPE-2(right) mice. Scale bar, 20 µm. g Western blot analysis of H3K27me3 in TP, TPE-1, and TPE-2 gastric tumor organoids. Representative blot ( g ) of n = 2 biological replicates. h Representative H&E, P40, CK14, CK5/6, and CK5 IHC staining of TP (top), TPE-1 (middle), and TPE-2(bottom) tumor section from mice. Scale bar, 50 µm. i Statistical graphs of keratin pearl area percentages in tumor tissues from TP, TPE-1, and TPE-2 mice ( n = 4 mice). j Representative bright-field (left) and red-fluorescence(right) images of liver from TP (top) and TPE (bottom) tumor-bearing mice. Scale bars, 2 mm(top) and 1 mm(bottom). k H&E staining of liver sections from TP (top) and TPE (bottom) tumor-bearing mice. Scale bar, 50 µm. l Representative IHC staining of CAS9, CK14, CK5/6 and P40 in liver sections from TPE tumor-bearing mice. Scale bar, 50 µm. Data are shown as means ± SD, p -value was determined by unpaired two-tailed t test( c , i ). The samples derive from the same experiment, and that gels were processed in parallel for quantitative comparisons (e.g.,). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were H3K27me3 (Abclonal, A2363,1:200), Ezh2 (D2C9) XP® Rabbit mAb (Cell Signaling Technology, 5246S, 1:50 for IHC, 1:1000 for IF), Anti-AP2 gamma/TFAP2C antibody [3B5] (Abcam, ab110635,1:50 for IHC, 1:500 for IF), Cytokeratin 5Monoclonal antibody(2C2) (Invitrogen, MA5-17057, 1:500 for IHC, 1:1000 for IF), Anti-Cytokeratin 14 antibody (Abcam, ab7800, 1:500), Cas9 (S. pyogenes) (E7M1H) XP® Rabbit mAb (Cell Signaling Technology, 19526S, 1:400), Anti-Cytokeratin 7 antibody [ EPR17078 ] - Cytoskeleton Marker (Abcam, ab181598, 1:4000), CK20 (Abcam, ab53120,1:100), anti-P40-DeltaNp63antibody [EPR17863-47] (Abcam, ab203826, 1:1000 for IHC, 1:50 for IF), Cytokeratin 5/6 Monoclonal antibody (Thermo Fisher Scientific, MA5-12429, 1:200), Anti-MUC1 antibody [EP1024Y] (Abcam, ab45167, 1:400), MUC5AC monoclonal antibody (45M1) (Thermo Fisher Scientific, MA5-12178, 1:400).

Techniques: Fluorescence, Transduction, Immunohistochemistry, Western Blot, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii

doi: 10.1038/s41598-020-78968-x

Figure Lengend Snippet: Translocation of cell penetrating peptide pVEC and ribonucleoprotein. ( a ) The confocal microscopy image for C. reinhardtii to investigate the translocation of pVEC (FITC) and RNP(Cy3). Cas9 and sgRNA were incubated for 30 min before treatment, and mixed with a cell sample. pVEC was added to the cell sample containing RNP and incubated for 20 min. After washing including trypsin treatment, cells were observed under confocal microscopy. White scale bar represent 10 μm. ( b ) Translocation of pVEC-FITC. The washed sample was resuspended in 200ul of TAP media and its fluorescent intensity at 511 nm was measured by a spectrofluorophotometer with excitation wavelength of 475 nm ( c ) Treatment of endocytosis inhibitor. The fluorescent intensity was measured at 511 nm and 570 nm for FITC and Cy3 respectively. Sodium azide (SA) was treated for 1 h with the final concentration of 10 mM. The data represent the average of n = 3 replicate experiments. Standard deviation bars are shown. **Significantly different (Student’s t-test, p < 0.05) and *Significantly not different (Student’s t-test, p > 0.05).

Article Snippet: Both wild-type Cas9 without NLS (Cas9 Nuclease, M0386M, NEB, US) and Cas9 with NLS (EnGen Cas9 NLS, M0646M, NEB, US) proteins were purchased from New England BioLabs. sgRNA was synthesized using T7 in-vitro transcription according to the protocol provided by the manufacturer (NEB).

Techniques: Translocation Assay, Confocal Microscopy, Incubation, Concentration Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii

doi: 10.1038/s41598-020-78968-x

Figure Lengend Snippet: Delivery efficiency of RNP and Cytotoxicity of Cas9 and ribonucleoprotein. ( a ) The cellular uptake efficiency of Cy3-RNP into wild-type C. reinhardtii measured by a flow cytometer according to the concentration of pVEC. The ratio of the quantum yield of wild-type C. reinhardtii according to the concentration of pVEC. After incubation of samples for one hour under dark as a dark adaptation, quantum yield was measured by AquaPen-C APA100. The ratio of the quantum yield was calculated by dividing the QY of the experimental group by the QY of the control group. The data represent the average of n = 3 replicate experiments. Standard deviation bars are shown. **Significantly different (Student’s t-test, p < 0.05). *Significantly not different (Student’s t-test, p > 0.05). ( b ) The recovery of algal cell on agar plate after treatment. Relative protein amount 1 represents 10ug of protein (Cas9 and alcohol dehydrogenase). 600 cells were spread and counted. The final concentration of pVEC for delivery of protein was 8uM. **Significantly different (Student’s t-test, p < 0.05). ( c ) The quantum yield for algal cell depends on time. Relative amount 1 of protein was used. The quantum yield was measured by AquaPen-C APA100. The data represent the average of n = 3 replicate experiments. Standard deviation bars are not shown for legibility.

Article Snippet: Both wild-type Cas9 without NLS (Cas9 Nuclease, M0386M, NEB, US) and Cas9 with NLS (EnGen Cas9 NLS, M0646M, NEB, US) proteins were purchased from New England BioLabs. sgRNA was synthesized using T7 in-vitro transcription according to the protocol provided by the manufacturer (NEB).

Techniques: Flow Cytometry, Concentration Assay, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii

doi: 10.1038/s41598-020-78968-x

Figure Lengend Snippet: Gene disruption of Maa7 responsible for tryptophan synthase beta unit (TSB) and gene disruption of FKB12. ( a ) In-vitro cleavage assay. PCR amplicon having a size of ~ 600 bp was cleaved into two fragments having size of 350 bp and 250 bp. Template DNA of 100 ng was used for the assay and 600 ng of Cas9 and 500 ng of sgRNA were used for in-vitro cleavage assay. Detailed methodology was described in the method part. ( b ) The Indel mutation cases for in-vivo genome editing experiments. The PAM site is shown in green, and the cleavage point by sgRNA is shown as a red arrow. ( c ) Indel mutation efficiency of C. reinhardtii subspecies CC124, cell wall-less mutant CC400 and UVM11. For cell wall-less mutant, a starch-embedding method was used for spreading. ( d ) Protein(FITC-BSA) delivery into C. reinhardtii subspecies by 8 μM of pVEC. Fluorescent intensity of FITC at 511 nm was measured. The washed sample was resuspended in 200ul of TAP media and its fluorescent intensity at 511 nm was measured by a spectrofluorophotometer with excitation wavelength of 475 nm. ( e ) Description of FKB 12 locus. Underlined sequences refer to CDS, green-sequence refer PAM site, and red-sequence refer sgRNA target site. ( f ) In-vitro cleavage assay using PCR amplicon of 600 bp. ( g ) Indel mutation cases for in-vivo genome editing experiments. ( h ) Insertion of in-vitro transcription DNA template. The data represent the average of n = 5 replicate experiments. Standard deviation bars are shown. **Significantly different (Student’s t-test, p < 0.05) and *Significantly not different (Student’s t-test, p > 0.05).

Article Snippet: Both wild-type Cas9 without NLS (Cas9 Nuclease, M0386M, NEB, US) and Cas9 with NLS (EnGen Cas9 NLS, M0646M, NEB, US) proteins were purchased from New England BioLabs. sgRNA was synthesized using T7 in-vitro transcription according to the protocol provided by the manufacturer (NEB).

Techniques: In Vitro, Cleavage Assay, Amplification, Mutagenesis, In Vivo, Sequencing, Standard Deviation

Journal: Neoplasia (New York, N.Y.)

Article Title: Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma

doi: 10.1016/j.neo.2025.101139

Figure Lengend Snippet: Generation of primary DMGs in mice using CRISPR/Cas9. (A) Post-natal day 3-5 NNC mice were intracranially injected with virus-secreting DF1 chicken fibroblast cells transfected with RCAS-Trp53-gRNA-BFP and RCAS-Cntl-gRNA-PDGFB plasmids. (B) Kaplan-Meier survival analysis of mice that received and did not receive intracranial injections of virus-secreting fibroblast cells on post-natal day 3-5. P-value for log-rank test comparison between groups. (C) Whole mount (top) and H&E slides showing tumor (bottom left) and non-tumor (bottom right) of NNC mice. Scale bar for whole mount H& E = 2 mm. Scale bar for H& E = 50 µm; images taken at 40x. (D) IHC images of mouse DMGs in NNC mice incubated with anti-Ki67, anti-HA, anti-Cas9, anti-GFP, or anti-p53 antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x. (E) IHC images of non-tumor brain tissue in NNC mice incubated with anti-Ki67, anti-HA, anti-Cas9, anti-GFP, or anti-p53 antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x.

Article Snippet: Antibodies used were rabbit monoclonal Cas9 primary antibody (#ab189380), rabbit monoclonal GFP primary antibody (#ab183734), rabbit monoclonal HA-Tag primary antibody (Cell Signaling #3724), rabbit polyclonal Ki67 primary antibody (#ab15580), rabbit polyclonal p53 primary antibody (#NCL-l-P53CM5P), and rabbit polyclonal PTEN primary antibody (#AF847).

Techniques: CRISPR, Injection, Virus, Transfection, Comparison, Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma

doi: 10.1016/j.neo.2025.101139

Figure Lengend Snippet: Two approaches to generate tumors containing CRISPR/Cas9 perturbations. (A) On the left, Cre-recombinase is expressed from an endogenous Nestin-Cre allele in the germline to drive Cas9 expression in all Nestin+ neural stem cells (NNC mice), allowing the use of only 2 viral constructs. On the right, Cre is delivered via a third retroviral construct (NC mice). (B) Anti-Cas9 IHC for NNC mice brain tissue. Scale bar for whole mount IHC = 2 mm. Scale bar for IHC of tumor and non-tumor tissue = 50 µm; images taken at 40x. (C) Anti-Cas9 IHC for NC mice brain tissue. Scale bar for whole mount IHC = 2 mm. Scale bar for IHC of tumor and non-tumor tissue = 50 µm; images taken at 40x. (D) Tumor penetrance and median time to tumor formation for NNC and NC mice.

Article Snippet: Antibodies used were rabbit monoclonal Cas9 primary antibody (#ab189380), rabbit monoclonal GFP primary antibody (#ab183734), rabbit monoclonal HA-Tag primary antibody (Cell Signaling #3724), rabbit polyclonal Ki67 primary antibody (#ab15580), rabbit polyclonal p53 primary antibody (#NCL-l-P53CM5P), and rabbit polyclonal PTEN primary antibody (#AF847).

Techniques: CRISPR, Expressing, Construct, Retroviral

Journal: Neoplasia (New York, N.Y.)

Article Title: Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma

doi: 10.1016/j.neo.2025.101139

Figure Lengend Snippet: Pooled in vivo CRISPR experiment. (A) Post-natal day 3-5 NNC mice were intracranially injected with virus-secreting DF1 chicken fibroblast cells transfected with RCAS-gRNA-PDGFB plasmids targeting Atm, Cdkn2a, Pten, and Trp53 respectively. (B) Kaplan-Meier analysis of mice monitored for survival following injection (mice analyzed in are shown for reference, P-value for log-rank test shown). (C) Whole mount (top) and H&E slides showing tumor (bottom left) and non-tumor (bottom right) of NNC mice. Scale bar for whole mount H& E = 2 mm. Scale bar for H& E = 50 µm; images taken at 40x. (D) IHC images of NNC mouse midline gliomas incubated with anti-Ki67, anti-Cas9, anti-p53, and anti-Pten antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x. (E) IHC images of NNC mouse non-tumor brain tissue incubated with anti-Ki67, anti-Cas9, anti-p53, and anti-Pten antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x.

Article Snippet: Antibodies used were rabbit monoclonal Cas9 primary antibody (#ab189380), rabbit monoclonal GFP primary antibody (#ab183734), rabbit monoclonal HA-Tag primary antibody (Cell Signaling #3724), rabbit polyclonal Ki67 primary antibody (#ab15580), rabbit polyclonal p53 primary antibody (#NCL-l-P53CM5P), and rabbit polyclonal PTEN primary antibody (#AF847).

Techniques: In Vivo, CRISPR, Injection, Virus, Transfection, Incubation

Journal: bioRxiv

Article Title: Prime editing of the β 1 adrenoceptor in the brain reprograms mouse behavior

doi: 10.1101/2023.05.19.541410

Figure Lengend Snippet: ( a ) Schematic representation of AAV designs used in vivo and their corresponding lengths in kilobase pairs (kbp, including ITRs) for neuron-specific expression of PEmax or PE3bmax. Constructs are not depicted to scale. ( b ) Schematic representation of the experimental setup and timeline. ( c ) In vivo prime editing and indel rates of different AAV vector designs in mouse cortices at 5 weeks, 10 weeks and 6 months post-injection. ( d ) Editing and indel rates at the Adrb1 (AAV-PE3bmax-nT and W3-synth-cT) and Dnmt1 (AAV-PEmax-nT and noW3-bGH-cT) locus in different brain regions at 10 weeks post-injection. ( e ) Frequency of Adrb1 and Dnmt1 edits in various other tissues in saline-and phsyn-PE-treated animals at 6 months post injection. Animals were treated with the same AAV preparations as in (d). Skeletal muscle tissue was isolated from the quadriceps femoris. ( f ) Editing rates at the Dnmt1 locus in mouse cortices (5 and 10 weeks post-injection), liver, and heart (10 weeks post-injection) after ICV injection. Animals were treated with PEmax-noW3-synth-nT and noW3-synth-cT under the control of the ubiquitous Cbh promoter . ( g ) Comparison of editing and indel rates at the Adrb1 locus for PEmax complexed with epegRNA1 or epegRNA1-SM CTT (with and without a PE3b-ngRNA) at 5 weeks. Animals were treated with AAV-PEmax-nT and AAV-W3-synth-cT. Data are displayed as means±s.d. of at least three animals. Each data point represents one animal. ITR, inverted terminal repeat; nT/cT, N-/C-terminal PEmax AAV vector; phsyn, human synapsin promoter; NLS, nuclear localization signal; n Sp Cas9, Sp Cas9 nickase; M-MLV, Moloney Murine Leukemia virus; W3, woodchuck hepatitis virus post-transcriptional regulatory element; hU6/mU6, human/mouse U6 promoter; SV40, Simian virus 40; pA, polyA signal; synth, synthetic polyA signal; bGH, bovine growth hormone polyA signal; kbp, kilobasepairs; vg, vector genomes; wks, weeks; m, months; SM, silent mutation; pCbh, truncated chimeric CMV/chicken-β-actin hybrid promoter.

Article Snippet: Sections were blocked in PBS supplemented with 2% normal donkey serum (cat. no. ab7475, abcam) and 0.3% Triton X-100 (Sigma-Aldrich) for 1 h. Brain sections were incubated with primary antibodies overnight at 4°C (mouse-NeuN, 1:500, abcam ab177487; rabbit-Cas9, 1:1,000, Cell Signaling clone D8Y4K; chicken-GFAP, 1:1’500, abcam ab95231).

Techniques: In Vivo, Expressing, Construct, Plasmid Preparation, Injection, Saline, Isolation, Control, Comparison, Virus, Mutagenesis