sequence encoding fkbp12f36v ha 2a mcherry  (Addgene inc)

Journal: bioRxiv

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1101/2025.05.08.652299

Figure Lengend Snippet: (a) Translation efficiency (log2 (Ribo-seq counts/RNA-seq counts)) negatively correlates with mRNA enrichment in P-bodies in naïve and primed mouse ES cells. Ribosome profiling data from , Pearson correlation test. (b) A schematic showing CRISPR-Cas9-based homozygous insertion of FKBP12F36V-HA-P2A-mCherry sequence in place of the stop codon of the endogenous Ddx6 allele. (c) Representative intracellular flow cytometry plots for DDX6 in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. (d) Representative IF imaging of EDC4 puncta (red) in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. Nuclei were counterstained with DAPI (blue) (scale: 10μm). (e) P-body number in Ddx6- FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. DMSO (n=70 cells), 3 hour-dTAG13 (n=70 cells), 6 hour-dTAG13 (n=70 cells), 9 hour-dTAG13 (n=70 cells), unpaired Student’s t-test, mean ± s.d., ****: p <0.0001. (f) Cumulative distribution function (CDF) plot showing ribosome occupancy (log2 FC) of P-body enriched and P-body-depleted mRNAs for untreated (DMSO) vs. dTAG-13 treated (6hrs) Ddx6- FKBP12 F36V GFP-LSM14A mouse naïve ES cells, ks-test. (g) Box plots showing the change in ribosome occupancy of all P-body enriched genes compared to all other genes. Unpaired t-test, mean ± s.d, padj (Holm’s method). (h) Normalized Enrichment Score (NES) of gene sets from (2C) , (Naïve) , and (Primed) , p<0.05. (i) Box plots showing the change in ribosome occupancy of P-body enriched 2C-related genes compared to non-P-body enriched 2C genes. Unpaired t-test, mean ± s.d, padj (Holm’s method). (j) Box plots showing the change in protein levels of all P-body enriched genes compared to P-body depleted genes, after 1 day and 3 days of dTAG-13 treatment. Unpaired t-test, mean ± s.d, padj (Holm’s method) (* p < 0.05, ** p < 0.01, **** p < 0.0001) (k) Box plots showing the change in protein levels of P-body enriched 2C-related genes compared to P-body depleted genes, after 1 day and 3 days of dTAG-13 treatment. Unpaired t-test, mean ± s.d, padj (Holm’s method). (l) Heatmap showing protein levels of 2C-related genes after 1 day and 3 days of dTAG-13 treatment compared to control samples.

Article Snippet: To introduce a sequence encoding FKBP12F36V-HA-2A-mCherry in place of the endogenous Ddx6 stop codon, a first donor plasmid was created by integrating two 200 bp homology arms specific to the Ddx6 gene into the pNQL004-SOX2-FKBPV-HA2-P2A-mCherry targeting construct (Addgene #175552).

Techniques: RNA Sequencing, CRISPR, Sequencing, Flow Cytometry, Imaging, Control

Journal: bioRxiv

Article Title: GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm

doi: 10.1101/2025.04.28.650944

Figure Lengend Snippet: (A) Schematic diagram of viral vector (AAV- U6-gGabrb1,2,3-EF1α-DIO-mCherry or AAV- U6-gControl-EF1α-DIO-mCherry ) injection at the SCN in Rosa26-CAG-LSL-SpCas9-2A-EGFP; Vip-ires-Cre mice to generate control or Vip-GABA A R −/− mice for locomotor activity recording. (B) A representative coronal section of mice with SpCas9-2A-EGFP and gRNA-DIO-mCherry expression in SCN VIP neurons. Green, SpCas9-EGFP; magenta, mCherry. (C) Representative locomotor activity of control and Vip-GABA A R −/− mice (home-cage activity). Gray shading indicates the time when the lights were off. (D) Averaged daily profile of locomotor activity in LD (left) or DD (right). (E) Average locomotor activity counts per 6-hour interval in LD (left) or DD (right). (F) Activity time of locomotor activity rhythm in LD (left) or in DD (right). Blue, Control; red, Vip-GABA A R −/− . Values are mean ± SEM. n = 8 for Control, n = 16 for Vip-GABA A R −/− mice. *P < 0.05; **P < 0.01 by two-way repeated measures ANOVA post-hoc two-tailed Student’s t-test with Bonferroni correction (E), or by two-tailed Student t tests (F).

Article Snippet: Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5’-agtacgcgTCGAGCATGCTCGAGAATGG-3’ and 5’-agtacgcgtCGGGTACCCCATTTGTCTGC-3’, and cloned into the MluI site of pAAV-EF1a-DIO-mCherry (a gift from Dr. Bryan Roth) as described previously [ ]. pAAV-U6-gGabrb2-EF1α-DIO-mCherry happened to contain two copies of the amplified fragment. pAAV-U6-gControl-EF1α-DIO-mCherry contains spacer sequences from pX333 (Addgene plasmid #64073, a gift from Dr. Andrea Ventura) instead of gRNA sequences for Gabrb3 genes. pAAV - CAG-FLEX-ChrimsonR-mCherry was described previously (9).

Techniques: Plasmid Preparation, Injection, Control, Activity Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm

doi: 10.1101/2025.04.28.650944

Figure Lengend Snippet: (A) Schematic diagram of viral vector (AAV- CAG-FLEX-jGCaMP7s and AAV- U6-gGabrb1,2,3-EF1α-DIO-mCherry or AAV- U6-gControl-EF1α-DIO-mCherry ) injection and optical fiber implantation at the SCN in Rosa26-CAG-LSL-SpCas9-2A-EGFP; Vip-ires-Cre mice to generate control or Vip-GABA A R −/− mice for fiber photometry recording. (B) A representative coronal section of mice with jGCaMP7s expression and gRNA-DIO-mCherry expression in SCN VIP neurons. A white dotted square shows the estimated position of the implanted optical fiber. Green, jGCaMP7s; magenta, mCherry. (C) Representative plots of the in vivo jGCaMP7s signal of SCN VIP neurons (green) overlaid with locomotor activity (home-cage activity) (black) in actograms. Control (Left) and Vip-GABA A R −/− (Right) mice were initially housed in LD (LD1 to LD7) and then in DD (DD1 to DD15). Gray shading indicates the time when the lights were off. (D) Plots of locomotor activity onset (black), activity offset (gray), VIP-Ca 2+ onset (green), VIP-Ca 2+ offset (light green), and VIP-Ca 2+ midpoint (magenta) of mean ± SEM (left column) and individual (right column) in control and Vip-GABA A R -/- mice. (E) Normalized VIP-Ca 2+ daily rhythm profiles in LD (LD3-7, top) or in DD (DD5-14,middle and bottom). In DD, circadian time 12 was determined as the onset of locomotor activity and VIP-Ca 2+ circadian time 0 was determined as the onset of VIP-Ca 2+ . (F) High VIP-Ca 2+ duration in LD (LD3-7, left) or in DD (DD5-14, right). (G) High VIP-Ca 2+ duration in LD (LD3-7, left) or in DD (DD5-14, right) calculated with 20% amplitude of the VIP-Ca 2+ profile as the threshold to determine the Ca 2+ onset and offset. Values are mean ± SEM. n = 7 for control, n = 5 for Vip-GABA A R −/− mice. *P < 0.05; **P < 0.01 by two-tailed Student t tests.

Article Snippet: Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5’-agtacgcgTCGAGCATGCTCGAGAATGG-3’ and 5’-agtacgcgtCGGGTACCCCATTTGTCTGC-3’, and cloned into the MluI site of pAAV-EF1a-DIO-mCherry (a gift from Dr. Bryan Roth) as described previously [ ]. pAAV-U6-gGabrb2-EF1α-DIO-mCherry happened to contain two copies of the amplified fragment. pAAV-U6-gControl-EF1α-DIO-mCherry contains spacer sequences from pX333 (Addgene plasmid #64073, a gift from Dr. Andrea Ventura) instead of gRNA sequences for Gabrb3 genes. pAAV - CAG-FLEX-ChrimsonR-mCherry was described previously (9).

Techniques: Plasmid Preparation, Injection, Control, Expressing, In Vivo, Activity Assay, Two Tailed Test