Journal: Journal of the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 1. The epigenetic factors DNMT1 and MeCP2 and the transcriptional factor REST are involved in the negative modulation of Ncx1 mRNA and protein expression. A and B, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y cells transfected for 48 hours with siDNMT1 or siMeCP2 at a final concentration of 50 nM. An siCTL was used as scrambled. *P≤0.05 vs siCTL by Student’s t test (n=3). C, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hours with siCTL, siDNMT1, siMeCP2, and siREST. *P≤0.05 vs siCTL by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). D and E, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y transfected for 48 hours with vector overexpressing DNMT1 or MeCP2. pcDNA3.1 vector was used as EV. *P≤0.05 vs EV by Student’s t test (n=3). F, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hour with EV, DNMT1, MeCP2, or REST plasmids. *P≤0.05 vs EV by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). CTL indicates control; DNMT1, DNA-methyltransferase-1; EV, empty vector; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; si, small interfering; and siCTL, control siRNA.

Article Snippet: Chromatin lysates were incubated overnight with 3 μg of antibody for DNMT1 (Mouse mAb, cod: sc- 271 729 Santa Cruz Biotechnology, dilution 1:500), MeCP2 (Rabbit mAb, cod: 3456 Cell Signaling, dilution 1:1000), REST, HDAC1, and HDAC2.3 Normal rabbit IgG was used as negative control.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Concentration Assay, Plasmid Preparation, Control, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 4. DNMT1 and MeCP2 bind Ht, but not Br promoter sequence, in the temporoparietal cortex 24 hours after tMCAO. Chromatin immunoprecipitation with anti-DNMT1 (A), anti-MeCP2 (B) anti-REST (C), anti-HDAC1 (D), anti-HDAC2 (E), antibodies followed by qPCR of Ncx1 brain promoter (Br) (white columns) and Ncx1 heart promoter (Ht) (gray columns) in peri-ischemic cortex of mice euthanized 12 or 24 hours after tMCAO. IgG was used as negative control. *P≤0.05 vs Sham immunopreciptated with IgG antibody by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3). DNMT1 indicates DNA-methyltransferase-1; HDAC, histone deacetylase; IP, immunoprecipitation; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; and tMCAO, transient middle cerebral artery occlusion.

Article Snippet: Chromatin lysates were incubated overnight with 3 μg of antibody for DNMT1 (Mouse mAb, cod: sc- 271 729 Santa Cruz Biotechnology, dilution 1:500), MeCP2 (Rabbit mAb, cod: 3456 Cell Signaling, dilution 1:1000), REST, HDAC1, and HDAC2.3 Normal rabbit IgG was used as negative control.

Techniques: Sequencing, Chromatin Immunoprecipitation, Negative Control, Histone Deacetylase Assay, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Alterations of Gene Expression and Glutamate Clearance in Astrocytes Derived from an MeCP2-Null Mouse Model of Rett Syndrome

doi: 10.1371/journal.pone.0035354

Figure Lengend Snippet: A. Expression of astroglial genes in primary cultured cortical astrocytes. Semi-quantitative RT-PCR analysis of Mecp2 and astroglial genes was performed in wild-type (white column) and MeCP2-null (gray column) astrocytes. Mecp2 e1 and e2 were detectable in the wild-type astrocytes. The lower graphs show that the GFAP/HPRT or S100β/HPRT expression ratio in each genotype was normalized against the level in control astrocytes. Bars represent the means ± standard errors (SE) of samples from three independent experiments (*p<0.05). The expression of astroglial markers was significantly upregulated by MeCP2 deficiency. B. Expression of MeCP2 in the primary cultured cortical astrocytes. The astrocytes were immunostained with MeCP2 (green) and GFAP (red) as glial-specific astrocytic markers. Scale bars indicate 50 µm.

Article Snippet: Cultures were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.05% Triton-X 100 for 5 min. After blocking of nonspecific binding sites with 10% nonfat dry milk in PBS for 1 h, cultures were immunocytochemically stained using antibodies against MeCP2 (anti-MeCP2 polyclonal antibody, MILLIPORE, Temecula, CA, USA; anti-MeCP2 monoclonal antibody, G-6, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), β-tubulin type III (TuJ, Sigma-Aldrich, Inc., St. Louis, Missouri), or glial fibrillary acidic protein (GFAP) (anti-GFAP polyclonal antibody, G9269; anti-GFAP monoclonal antibody, G3893, Sigma-Aldrich, Inc., St. Louis, Missouri), followed by secondary fluorescent antibodies as described previously .

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Control

Journal: PLoS ONE

Article Title: Alterations of Gene Expression and Glutamate Clearance in Astrocytes Derived from an MeCP2-Null Mouse Model of Rett Syndrome

doi: 10.1371/journal.pone.0035354

Figure Lengend Snippet: A. Comparison of cell growth in wild-type and MeCP2-deficient astrocytes. As passage number increased, cell growth rate decreased dramatically in both strains of astrocytes. There was no significant difference in growth rate between the control and MeCP2-null astrocyte cultures. B. Quantification of BrdU-incorporating cells in control and MeCP2-null astrocytes. Astrocytes were cultured for 24 h and incubated with BrdU for 2 h. The graph shows the percentage of BrdU-incorporating cells in the control (white column) and MeCP2-deficient (gray column) astrocytes 2 h after BrdU exposure. The number of BrdU-incorporating cells is expressed as a percentage of the total number of Hoechst-stained cells . Bars represent the means ± SE of the samples from four independent experiments. The ratio of BrdU-incorporating cells is similar in astrocytes taken from both control and MeCP2-null strains. C–E. Comparison of effects of various neurotoxins ( C, H 2 O 2 ; D, NH 4 Cl; E, Glutamate) on control and MeCP2-null astrocytes. The graph shows the percentage of viability in the control (white column) and MeCP2-deficient (gray column) astrocytes after neurotoxin treatment at the indicated concentrations. Bars represent the means ± SE of samples from three independent experiments. The glial cultures showed no difference in viability between the control and MeCP2-null strains.

Article Snippet: Cultures were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.05% Triton-X 100 for 5 min. After blocking of nonspecific binding sites with 10% nonfat dry milk in PBS for 1 h, cultures were immunocytochemically stained using antibodies against MeCP2 (anti-MeCP2 polyclonal antibody, MILLIPORE, Temecula, CA, USA; anti-MeCP2 monoclonal antibody, G-6, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), β-tubulin type III (TuJ, Sigma-Aldrich, Inc., St. Louis, Missouri), or glial fibrillary acidic protein (GFAP) (anti-GFAP polyclonal antibody, G9269; anti-GFAP monoclonal antibody, G3893, Sigma-Aldrich, Inc., St. Louis, Missouri), followed by secondary fluorescent antibodies as described previously .

Techniques: Comparison, Control, Cell Culture, Incubation, Staining

Journal: PLoS ONE

Article Title: Alterations of Gene Expression and Glutamate Clearance in Astrocytes Derived from an MeCP2-Null Mouse Model of Rett Syndrome

doi: 10.1371/journal.pone.0035354

Figure Lengend Snippet: A–C. Effects of Glu on Glu clearance-related genes in wild-type (white column) and MeCP2-null (gray column) astrocytes. Semi-quantitative RT-PCR analysis of Glu clearance-related genes, EAAT1 ( A ), EAAT2 ( B ), and GS ( C ), was performed in the control and MeCP2-null astrocytes 12 or 24 h after treatment with 1.0 mM Glu. The bands corresponding to PCR products were quantified by densitometry, normalized against HPRT levels, and expressed as % of controls (equals 100%). Bars represent the means ± SE of samples from 3–4 independent experiments (*p<0.05, **p<0.01). D–F. Comparison of the effects of Glu on EAAT1, EAAT2 or GS expression in the control and MeCP2-null astrocytes. The ratio of EAAT1/HPRT ( D ), EAAT2/HPRT ( E ) or GS/HPRT ( F ) in each treatment group was normalized against that of the non-treated astrocytes from each group. Bars represent the means ± SE of samples from 3–5 independent experiments (*p<0.05). Numbers in each column indicate the total number of samples analyzed.

Article Snippet: Cultures were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.05% Triton-X 100 for 5 min. After blocking of nonspecific binding sites with 10% nonfat dry milk in PBS for 1 h, cultures were immunocytochemically stained using antibodies against MeCP2 (anti-MeCP2 polyclonal antibody, MILLIPORE, Temecula, CA, USA; anti-MeCP2 monoclonal antibody, G-6, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), β-tubulin type III (TuJ, Sigma-Aldrich, Inc., St. Louis, Missouri), or glial fibrillary acidic protein (GFAP) (anti-GFAP polyclonal antibody, G9269; anti-GFAP monoclonal antibody, G3893, Sigma-Aldrich, Inc., St. Louis, Missouri), followed by secondary fluorescent antibodies as described previously .

Techniques: Quantitative RT-PCR, Control, Comparison, Expressing

Journal: PLoS ONE

Article Title: Alterations of Gene Expression and Glutamate Clearance in Astrocytes Derived from an MeCP2-Null Mouse Model of Rett Syndrome

doi: 10.1371/journal.pone.0035354

Figure Lengend Snippet: A. Time-dependent reduction of extracelluar Glu concentration in wild-type (white column) and MeCP2-null (gray column) astrocyte cultures. After treatment with 1.0 mM Glu, culture supernatant was collected at the indicated times for the determination of Glu concentration. The graph shows the concentration of Glu in control and MeCP2-null astrocyte culture medium. Bars represent the means ± SE of samples from three independent experiments (*p<0.05). B–D. Effects of inhibitors of glutamate transporters ( B, TBOA; C, DHKA; D, UCPH) on Glu clearance. Astrocytes were exposed to the indicated concentration of Glu transporter inhibitors, and then 0.1 mM Glu was added; culture supernatant was collected for the determination of Glu concentration at 2 h. The graphs show the clearance ratio upon treatment with each inhibitor. The clearance ratio in the indicated concentration groups was expressed by defining the control level (no inhibitor) as 1.0. Bars represent the means ± SE of samples from three independent experiments.

Article Snippet: Cultures were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.05% Triton-X 100 for 5 min. After blocking of nonspecific binding sites with 10% nonfat dry milk in PBS for 1 h, cultures were immunocytochemically stained using antibodies against MeCP2 (anti-MeCP2 polyclonal antibody, MILLIPORE, Temecula, CA, USA; anti-MeCP2 monoclonal antibody, G-6, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), β-tubulin type III (TuJ, Sigma-Aldrich, Inc., St. Louis, Missouri), or glial fibrillary acidic protein (GFAP) (anti-GFAP polyclonal antibody, G9269; anti-GFAP monoclonal antibody, G3893, Sigma-Aldrich, Inc., St. Louis, Missouri), followed by secondary fluorescent antibodies as described previously .

Techniques: Concentration Assay, Control

Journal: PLoS ONE

Article Title: Alterations of Gene Expression and Glutamate Clearance in Astrocytes Derived from an MeCP2-Null Mouse Model of Rett Syndrome

doi: 10.1371/journal.pone.0035354

Figure Lengend Snippet: A. Time-dependent expression of GS and EAAT1 proteins in wild-type and MeCP2-deficient astrocyte cultures. Astrocytes were treated with 1.0 mM Glu for 24 h, and subsequently analyzed for expression of GS and EAAT1 by Western blot analysis. Beta-actin protein levels were analyzed in the same way, as an internal control. B. The immunoreactive GS protein bands were quantified by densitometry, normalized against β-actin levels, and expressed as fold change relative to the controls (equals 1.0). Bars represent the means ± SE of samples from three independent experiments (*p<0.05, **p<0.01). Numbers in each column indicate the total number of samples analyzed. C. The immunoreactive EAAT1 protein bands were quantified by densitometry, normalized against β-actin levels, and expressed as % of controls (equals 100%). Bars represent the means ± SE of samples from three independent experiments (**p<0.01). D. Comparison of the effects of Glu on EAAT1 expression in wild-type and MeCP2-null astrocytes. The ratio of EAAT1/β-actin in each treatment group was normalized against that of the non-treated astrocytes from each group. Bars represent the means ± SE of samples from three independent experiments. Numbers in each column indicate the total number of samples analyzed.

Article Snippet: Cultures were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.05% Triton-X 100 for 5 min. After blocking of nonspecific binding sites with 10% nonfat dry milk in PBS for 1 h, cultures were immunocytochemically stained using antibodies against MeCP2 (anti-MeCP2 polyclonal antibody, MILLIPORE, Temecula, CA, USA; anti-MeCP2 monoclonal antibody, G-6, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), β-tubulin type III (TuJ, Sigma-Aldrich, Inc., St. Louis, Missouri), or glial fibrillary acidic protein (GFAP) (anti-GFAP polyclonal antibody, G9269; anti-GFAP monoclonal antibody, G3893, Sigma-Aldrich, Inc., St. Louis, Missouri), followed by secondary fluorescent antibodies as described previously .

Techniques: Expressing, Western Blot, Control, Comparison

Journal: PLoS ONE

Article Title: Alterations of Gene Expression and Glutamate Clearance in Astrocytes Derived from an MeCP2-Null Mouse Model of Rett Syndrome

doi: 10.1371/journal.pone.0035354

Figure Lengend Snippet: PCR primers.

Article Snippet: Cultures were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.05% Triton-X 100 for 5 min. After blocking of nonspecific binding sites with 10% nonfat dry milk in PBS for 1 h, cultures were immunocytochemically stained using antibodies against MeCP2 (anti-MeCP2 polyclonal antibody, MILLIPORE, Temecula, CA, USA; anti-MeCP2 monoclonal antibody, G-6, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), β-tubulin type III (TuJ, Sigma-Aldrich, Inc., St. Louis, Missouri), or glial fibrillary acidic protein (GFAP) (anti-GFAP polyclonal antibody, G9269; anti-GFAP monoclonal antibody, G3893, Sigma-Aldrich, Inc., St. Louis, Missouri), followed by secondary fluorescent antibodies as described previously .

Techniques:

Journal: Diabetes

Article Title: Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning.

doi: 10.2337/db19-0502

Figure Lengend Snippet: Fig. 1 Mecp2 is upregulated in WATs of obese humans/mice and during in vivo adipogenesis. (A-C) qPCR analysis (A), representative images (B) and western blots (left) with quantitative results (right) (C) of Mecp2 in eWAT of NC- or six-month HFD-fed male mice. (n = 5-6 per mouse group). (D) qPCR analysis of MECP2 in sWAT of human subjects (normal human subjects (n = 17)) and obese human subjects (n = 15). (E) Correlation between MECP2 and body mass index (BMI). (F-G) western blots (F; normal human subjects, n = 5) and obese human subjects (n = 7) and representative images (G; normal human subjects, n = 10) and obese human subjects (n = 12) of MECP2 in sWAT of human subjects. (H-K) Increased Mecp2 levels during white adipocyte differentiation in SVF and 3T3-L1 cells. The representative mRNA levels of indicated genes (H and J) and representative western blots (left) with quantitative results (right) of Pparγ1/2, Fabp4 and Mecp2 (I and K) during SVF and 3T3-L1 white differentiation, respectively. * P < 0.05; * * P < 0.01.

Article Snippet: To overexpress Mecp2 in 3T3-L1 cells, cells was transfected with plasmid pCMV6-mMecp2-Flag (Origene).

Techniques: In Vivo, Western Blot

Journal: Diabetes

Article Title: Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning.

doi: 10.2337/db19-0502

Figure Lengend Snippet: Fig. 6 Mecp2 negatively regulates Slpi level. (A) Volcanic map of several highly upregulated genes in iWAT of Mecp2Adi KO mice. (B and C) Slpi level in iWAT (B) and SVF or mature adipocytes of iWAT (C) of WT or

Article Snippet: To overexpress Mecp2 in 3T3-L1 cells, cells was transfected with plasmid pCMV6-mMecp2-Flag (Origene).

Techniques:

Journal: Diabetes

Article Title: Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning.

doi: 10.2337/db19-0502

Figure Lengend Snippet: Fig. 7 Mecp2 regulates browning by Slpi. (A) Experimental design and iWAT AAVs injection. (B) mRNA level of Slpi in iWAT injected with AAV-shSlpi or AAV-Scram. (C) Weights of iWAT. (D-E) Representative images of H&E/Ucp1 staining (D) and representative western blots (left) with quantitative results (right) of Ucp1 (E) in iWAT of WT or Mecp2Adi KO mice after 4◦C stress. (F) mRNA levels of thermogenic genes in iWAT of WT or Mecp2Adi KO mice after 4◦C stress. (G) serum Slpi level after iWAT pads of both flanks injected with AAV- shSlpi or AAV-Scram. n = 3-7 per group , * P < 0.05; * * P < 0.01.

Article Snippet: To overexpress Mecp2 in 3T3-L1 cells, cells was transfected with plasmid pCMV6-mMecp2-Flag (Origene).

Techniques: Injection, Staining, Western Blot

Journal: PNAS nexus

Article Title: Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

doi: 10.1093/pnasnexus/pgad170

Figure Lengend Snippet: Fig. 2. In vitro exposure of AAV copackaged Cas9 DNA with HTR2A-targeting gRNA leads to a decrease in spontaneous electrical activity of primary mouse cortical neurons. MEA analysis was performed in mouse cortical neurons following treatment with a mixture of AAV9 vectors containing AAV9– Mecp2–spCas9–sPA and AAV9–GFP–U6–mHtr2a–gRNA–ssODN at various concentrations indicated by the MOI. Neurons were treated at day 6, and MEA analyses were performed on day 14 (see the Methods section for details). A) Transduction was confirmed in primary cortical neurons by detecting GFP fluorescence. As shown, strong GFP fluorescence was observed at a MOI of 2 × 105, with the intensity decreasing significantly at 2 × 104, and 2 × 103 MOI, respectively. B) There are 16 electrodes in each well, and each panel represents the recording of the real-time signal of one selected electrode at different MOIs. The gray threshold lines represent the voltage of the baseline noise. If an electrode signal is beyond the threshold, it will be recorded as a spike. As the MOI increased, the number of spikes decreased significantly as compared with vehicle. C) Shown are continuous spike signals of all 16 electrodes within 50 s. Each row presents an electrode. The bursts are shown as vertical bars. A network of bursts is a coordinated cluster of spiking across multiple electrodes. There were network bursts in the vehicle and lowest MOI, but not at higher MOIs including 2 × 104 and 2 × 105. D–F) Quantification of MEA analysis showing the total number of spikes over the duration of the analysis D), the number of network bursts defined as a cluster of spikes across all electrodes E), and the synchrony index, which indicates a unitless measure of synchrony between 0 and 1. Values closer to 1 indicate higher synchrony. In all cases, exposure of neurons to 2 × 105 MOI led to a significant decrease in the number of spikes (83% decrease compared with vehicle controls, P = 0.0007, N = 3) D); in the number of bursts (68% decrease compared with vehicle controls, P = 0.014, N = 3) E); and a decrease in the synchrony index (34% decrease compared with vehicle controls, P = 0.0002, N = 3) F). G) and H) are identical in terms of experimental procedure as A–F), with the exception that genomic DNA was collected following treatments and next-generation targeted sequencing was performed as described in the Methods section. G) depicts the efficiency analysis of gene editing in a stacked column graph with the total number of reads (unmapped versus mapped) for each treatment group. The unmapped reads represent the counts of sequence that were inconsistent with the reference sequence (HTR2A gene). The mapped reads represent the counts of sequence that were consistent with the reference sequence. At the highest concentration, MOI = 2 × 105, a 41.4% gene editing efficiency was observed, followed by a 2% at MOI of 2 × 104. In H), indels within the target sequence of the HTR2A gene (149 bp) are shown with the top panel representing the insertion ratio, while the bottom pattern representing the deletion ratio. In both cases, the highest MOI concentration led to a sharp increase in indel formation within the target site. NGG in both graphs indicates the PAM sequence. AAV9 volumes of 40, 4, and 0.4 µL correspond to a MOI of 2 × 105, 2 × 104, and 2 × 103, respectively (see Table 1 of main text).

Article Snippet: The plasmid design for expression of spCas9 under the neuronal-specific promoter, MeCP2, was from Addgene (17).

Techniques: In Vitro, Activity Assay, Transduction, Fluorescence, Sequencing, Concentration Assay

Journal: PNAS nexus

Article Title: Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

doi: 10.1093/pnasnexus/pgad170

Figure Lengend Snippet: Fig. 3. Intranasal delivery of AAV9 vectors containing Cas9 and gRNA receptor leads to down-regulation of 5HT-2A receptor mRNA. A) Experimental workflow (see Methods for details). AAV9–Mecp2–spCas9–sPA and AAV9–GFP–U6–mHtr2a–gRNA–ssODN were synthesized and typical viral titers were on the order of 2.0 × 1013 GC/mL. The plasmid design for expression of spCas9 under the neuronal-specific promoter, MeCP2, was from Addgene (17). Separately, AAV9 vectors were constructed that contained the gRNA for the HTR2A gene with a GFP coexpression system. Both vectors were mixed at equal concentrations and delivered intranasally via a micropipette tip (∼2.0 × 1011 viral particles). B) Representative, low-field immunofluorescence sagittal image following treatment, fixation in formalin, and immunolabeling using a specific antibody against GFP (1:1,000). GFP-positive neurons positively transfected with gRNA were identified in most brain regions including the olfactory bulb (OB), cortex, cerebellum, and numerous subcortical areas including the IPN, a major connectome for stress-mediated pathways. C) Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of total brain RNA from frozen brain tissue in either vehicle controls (green bar) or CRISPR/Cas9 treated (bar labeled ‘treated’). Results display the relative change in expression. Real-time PCR results represent a total of N = 5 animals for each group performed in triplicate ± SEM. Asterisk denotes significant difference between the two groups, P ≤0.05. D) NGS analyses on-target effects in five individual mouse brain samples were analyzed and the most prevalent reads identified are shown. The profile mutations induced by HTR2A-targeting AAV-CRISPR/Cas9 revealed single base pair deletions indicated by the −1 symbol in adenine, which occurred in the exact positions for all five animals. E) The corresponding predicted amino acid sequences are presented for the two adenine indels with both mutations leading to a nonsense introduction of a premature stop codon. *Denotes single base pair deletion of adenine closest to the PAM sequnce. **Denotes single base pair deletion of adenine farthest from the PAM sequence. PAM, protospacer adjacent motif; WT, wild type.

Article Snippet: The plasmid design for expression of spCas9 under the neuronal-specific promoter, MeCP2, was from Addgene (17).

Techniques: Synthesized, Plasmid Preparation, Expressing, Construct, Immunofluorescence, Immunolabeling, Transfection, Extraction, CRISPR, Labeling, Real-time Polymerase Chain Reaction, Sequencing

Journal: Cell reports

Article Title: Systematic evaluation of retroviral LTRs as cis -regulatory elements in mouse embryos

doi: 10.1016/j.celrep.2024.113775

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The dCas9-HA-NLS-NLS-KRAB-NLS sequence was amplified from the plasmid PB-TRE-dCas9-KRAB-MeCP2 (addgene 122267) using PrimeSTAR Max2× premix (Takara).

Techniques: Virus, Recombinant, SYBR Green Assay, Protease Inhibitor, Immunoprecipitation, Transfection, cDNA Synthesis, Expressing, Plasmid Preparation, Software