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: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Schematic illustration of the design and workflow. ( A ) Engineering guide RNA to make it responsive to small molecules. The gRNA scaffold contains multiple well-defined structural motifs, including a repeat:anti-repeat duplex and three stem–loops. NCD and its hydrogen-bonding pattern to the introduced G-G mismatch sites are demonstrated. The structural unit of N-acyl-2-amino-1,8-naphthyridine contains three hydrogen-bonding groups that are fully complementary to that of G. ( B ) Illustration of interference of Cas9-mediated DNA cleavage by RNA-binding small molecules. The Cas9 complex with designer gRNAs still retains wild-type levels of activity, while the exposure of this complex to MBLs leads to evident inhibition of DNA cutting activities. The protospacer adjacent motif (PAM) is indicated. Red rectangles: 2-amino-1,8-naphthyridine.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: RNA Binding Assay, Activity Assay, Inhibition

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Rational gRNA engineering for switching CRISPR/Cas9. ( A ) Sequence and structure analysis of gRNA scaffold (tracrRNA demonstration). We have identified a total of three motifs with close resemblance with the units possessing affinity for MBL binding. ( B ) MBLs are used to introduce structural constraints into gRNAs through hydrogen bonding and stacking. Sequence modification sites are indicated in red. Red rectangles: 2-amino-1,8-naphthyridine.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: CRISPR, Sequencing, Binding Assay, Introduce, Modification

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of designer sgRNAs for switching CRISPR/Cas9 Reactions were performed as described in the Experimental Section. Uncleaved SLX4IP DNA (773 bp) cut to shorter cleavage fragments (441 bp and 332 bp) are demonstrated. All samples were tested in three biological replicates. Image of representative data is shown here. ( A ) Sanger sequencing analysis of selected sgRNAs. The sites for sequence modification are indicated. ( B ) The tolerance of Cas9 to each designer sgRNA. Lane 1: target control; lane 2: Cas9-only control; lane 3 contains original sg- SLX4IP ; lanes 4–10 contain designer sgRNAs harboring different MBL-binding units; lane 11: DNA marker (GeneRuler 100-bp DNA Ladder). ( C ) Responsiveness of designer sgRNAs to different MBLs. Lane 1: no Cas9 control; lanes 2–5 contain original sg- SLX4IP ; lanes 6–10, 11–15 contain sg- SLX4IP -S2c; lane 16: DNA marker. ( D ) The NCD-dependent inhibition of CRISPR/Cas9 with single-site variants. Lane 1: no Cas9 control; lanes 2–3 contain original sg- SLX4IP ; lanes 4–9 contain sg- SLX4IP -S1a; lanes 10–15 contain sg- SLX4IP -S1b; lanes 16–21 contain sg- SLX4IP -S1c; lane 22: DNA marker. ( E ) The NCD-dependent inhibition of CRISPR/Cas9 with multi-nucleotide variants. Lane 1: no Cas9 control; lanes 2–3 contain original sg- SLX4IP ; lanes 4–8 contain sg- SLX4IP -S2a; lanes 9–13 contain sg- SLX4IP -S2b; lanes 14–18 contain sg- SLX4IP -S2c; lanes 19–23 contain sg- SLX4IP -S3; lane 24: DNA marker.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: CRISPR, Sequencing, Modification, Binding Assay, Marker, Inhibition

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of designer tracrRNAs for switching CRISPR/Cas9 Reactions were performed as described in the Experimental Section. All samples were tested in three biological replicates. Image of representative data was shown here. ( A ) Illustration of MBL-responsive CRISPR/Cas9 with designer tracrRNAs. Red rectangles: 2-amino-1,8-naphthyridine. ( B ) The tolerance of Cas9 to each designer tracrRNA. Lane 1: target control; lane 2: Cas9-only control; lane 3 contains cr- SLX4IP and original tracrRNA; lanes 4–10 contain cr- SLX4IP and designer tracrRNAs harboring different MBL-binding units; lane 11: DNA marker (GeneRuler 100-bp DNA Ladder). ( C ) Dose-dependent response of the indicated tracrRNAs to each MBL. Lane 1: no Cas9 control; lanes 2–5 contain cr- SLX4IP and original tracrRNA; lanes 6–10, 11–15 contain cr- SLX4IP and tracrRNA-S2c; lane 16: DNA marker. ( D ) Effects of NCD on the function of tracrRNA and its single-site variants. Lane 1: no Cas9 control; lanes 2–3 contain cr- SLX4IP and original tracrRNA; lanes 4–9 contain cr- SLX4IP and tracrRNA-S1a; lanes 10–15 contain cr- SLX4IP and tracrRNA-S1b; lanes 16–21 contain cr- SLX4IP and tracrRNA-S1c; lane 22: DNA marker. ( E ) Effects of NCD on the function of tracrRNA and its multi-nucleotide variants. Lane 1: no Cas9 control; lanes 2–3 contain cr- SLX4IP and original tracrRNA; lanes 4–8 contain cr- SLX4IP and tracrRNA-S2a; lanes 9–13 contain cr- SLX4IP and tracrRNA-S2b; lanes 14–18 contain cr- SLX4IP and tracrRNA-S2c; lanes 19–23 contain cr- SLX4IP and tracrRNA-S3; lane 24: DNA marker.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: CRISPR, Binding Assay, Marker

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of designer sgRNAs in a stable Cas9-expressing cell line Cellular studies were performed using HeLa-OC cells as described in the Experimental Section. The treatment for each sample is indicated by the signs at the bottom of each lane. All samples were tested in three biological replicates. Image of representative data is shown here. ( A ) Editing of SLX4IP gene in HeLa-OC cells using the indicated sgRNAs. Lane 1: target control; lane 2: no sgRNA control; lane 3 contains original sg- SLX4IP ; lanes 4–10 contain designer sgRNAs harboring different MBL-binding units; lane 11: DNA marker (GeneRuler 100-bp DNA Ladder). ( B ) The effect of sequence modification on the function of sgRNAs in cells. ( C ) Ligand control of designer sgRNAs in HeLa-OC cells. Hela-OC cells were exposed to the NCD ligand for 24 h before being harvested for DNA cleaving activity assessments. Lane 1: target control; lanes 2–3: no sgRNA control; lanes 4–5 contain original sg- SLX4IP ; lanes 6–9 contain sg- SLX4IP -S1b; lanes 10–13 contain sg- SLX4IP -S1c; lanes 14–17 contain sg- SLX4IP -S2c; lane 18: DNA marker. ( D ) Bar graph shows the effect of NCD on the function of sgRNAs in HeLa-OC cells. In each group, the indel formation of NCD-treated cells were compared to that of mock-treated cells. P values less than 0.05 are given one asterisk, and P values <0.001 are given three asterisks. For (A) and (C), uncleaved SLX4IP DNA (773 bp) cut to shorter cleavage fragments (441 bp and 332 bp) are demonstrated. For (B) and (D), data represent the mean of three replicates and were shown as mean ± SEM.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: Expressing, Binding Assay, Marker, Sequencing, Modification, Activity Assay

Journal: Nucleic Acids Research

Article Title: Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing

doi: 10.1093/nar/gkac255

Figure Lengend Snippet: Ligand control of plasmid-based gene editing in human cells Cellular studies were performed as described in the Experimental Section. The plasmids and/or sgRNAs were delivered into HeLa cells before the treatment with NCD. Hela cells were exposed to the NCD ligand for 24 h before being harvested for DNA cleaving activity assessments. All samples were tested in three biological replicates. Image of representative data is shown here. ( A ) Ligand control of the hybrid system with IVT sgRNAs and Cas9-only plasmids. Lane 1: target control; lanes 2–3: no sgRNA control; lanes 4–5 contain PX165 and sg- SLX4IP ; lanes 6–9 contain PX165 and sg- SLX4IP -S1b; lanes 10–13 contain PX165 and sg- SLX4IP -S1c; lanes 14–17 contain PX165 and sg- SLX4IP -S2c; lane 18: DNA marker (GeneRuler 100-bp DNA Ladder). ( B ) Bar graph shows the effect of NCD on the function of the hybrid system (IVT sgRNAs and PX165). ( C ) Ligand control of all-in-one plasmids with MBL-binding units. Lane 1: target control; lanes 2–3: no plasmid control; lanes 4–5 contain PX459- SLX4IP ; lanes 6–9 contain PX459-S2c- SLX4IP ; lane 10: DNA marker. ( D ) Bar graph shows the effect of NCD on the function of all-in-one plasmids. For (A) and (C), uncleaved SLX4IP DNA (773 bp) cut to shorter cleavage fragments (441 bp and 332 bp) are demonstrated. For (B) and (D), the data are presented as the means ± SEM from three independent experiments. In each group, the indel formation of NCD-treated cells were compared to that of mock-treated cells. P values less than 0.05 are given one asterisk, P values <0.01 are given two asterisks, and P values <0.001 are given three asterisks.

Article Snippet: Cas9 nuclease, Streptococcus pyogenes (product# M0646), Bst DNA pol, Large Fragment (product# M0275), T4 DNA Ligase (product# M0202S), Ribonucleotide solution mix (NTPs, product# N0450) and deoxy-ribonucleoside triphosphates (dNTPs, product# N0446) were purchased from New England Biolabs (Ipswich, MA, USA).

Techniques: Plasmid Preparation, Activity Assay, Marker, Binding Assay

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

Journal: Infectious Disease Clinics of North America

Article Title: USE OF THE CLINICAL MICROBIOLOGY LABORATORY FOR THE DIAGNOSIS AND MANAGEMENT OF INFECTIOUS DISEASES RELATED TO THE ORAL CAVITY

doi: 10.1016/S0891-5520(05)70108-2

Figure Lengend Snippet: LABORATORY DETECTION OF ORAL MICROBIAL PATHOGENS

Article Snippet: In a national survey, the College of American Pathologists (CAP) sent 2692 laboratories a swab seeded with S. pyogenes to perform a rapid detection test with the kit used at their institution.

Techniques: Infection, Virus, Diagnostic Assay