lentiviral vector pgrna  (Addgene inc)

Journal: The Journal of Neuroscience

Article Title: Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation

doi: 10.1523/jneurosci.1453-23.2023

Figure Lengend Snippet: Figure 2. Artificially placing RPT6 at the Egr2 promoter drives expression after weak contextual fear conditioning in the CA1 region of male rats. A, Schematic of plasmid constructs, with the top showing construct of the dCas9-gRNA plasmid and the bottom showing our custom dCas9-RPT6-FLAG fusion plasmid. B, Immunofluorescent image showing 20× images of CRISPR gRNA expression (red; top left) and 3× FLAG expression (green; top right) in the CA1 region of the hippocampus 28 d after plasmid infusion. Merged images of 3× FLAG, CRISPR gRNA, and DAPI are shown at 20× magnification (bottom left) and 40× magnification (bottom right). C, Male rats received a bilateral infusion of either Egr2 gRNA alone (control) or Egr2 gRNA with the CRISPR- dCas9-RPT6-FLAG plasmid (Egr2 + dCas9-RPT6) into area CA1 of the dorsal hippocampus. After 28 d, rats were trained to a weak contextual fear conditioning, euthanized 1 h later, and then the CA1 region of the dorsal hippocampus was collected. D, Egr2 expression was increased in control trained and Egr2 + dCas9-RPT6 trained animals following weak training compared to control naïve animals. The magnitude of significance was larger in Egr2 + dCas9-RPT6 trained animals, as indicated by Tukey’s HSD post hoc test. E, Chromatin immunoprecipitation of RPT6 revealed decreased levels of RPT6 at the Egr2 promoter of control trained rats compared to control naïve rats, but no difference in RPT6 levels at the 3′ UTR region of Egr2. *p < 0.05, ***p < 0.001, ****p < 0.0001 from control naïve.

Article Snippet: The sequence (AGACCC GGGCGGTTGTCCAC) was inserted into the 293-T backbone and then cloned into the CRISPR gRNA vector (#44248 Addgene).

Techniques: Expressing, Plasmid Preparation, Construct, CRISPR, Control, Chromatin Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation

doi: 10.1523/jneurosci.1453-23.2023

Figure Lengend Snippet: Figure 3. The serine 120 (S120) codon is necessary in the CA1 region of male rats for RPT6 to bind DNA following context fear conditioning. A, The CRISPR-dCas13b-ADAR2DD RNA editing system was used to target the adenosine (bold) in the sequence coding for serine 120 (S120; highlighted blue). A 50 bp gRNA (indicated by underlined sequence) was generated against the RPT6-coding gene, Psmc5, mRNA sequence with a mismatch at the position 34 targeting adenosine. B, The gRNA plasmid targeting S120 with the dCas13b-ADAR2DD plasmid (Cas13-S120) or dCas13b-ADAR2DD plasmid alone (control) were transfected into rat B35 neuroblastoma cells and then collected 48 h later. Western blot analysis revealed that pRPT6-S120 levels normalized to total RPT6 were downregulated. In representative Western blot images, the top box is pRPT6-S120 levels, and the bottom box is total RPT6 levels. C, Male rats received a bilateral infusion of either gRNA targeting S120 and dCas13b-ADAR2DD plasmids (RPT6 + Cas13 trained) or dCas13b-ADAR2DD plasmid alone (control) into area CA1 of the dorsal hippocampus. After 28 d, rats were trained to standard contextual fear conditioning, euthanized 1 h later, and then the CA1 region of the dorsal hippocampus was collected. D, Altering S120 of RPT6 through dCas13 targeting did not impact performance during training. E, Egr2 expression was significantly different between all. Both control trained and RPT6 + Cas13 trained animals had increased expression compared to control naïve rats, and animals in the RPT6 + Cas13 trained group also had increased expression compared to control trained animals. F, Chromatin immunoprecipitation revealed increases in RPT6 bound to the 3′ UTR of Egr2 in control trained animals compared to control naïve and RPT6 + Cas13 trained animals. There were no significant differences in levels of phosphorylation at S120 of RPT6 (pRPT6-S120) or monoubiquitination of histone H2B at lysine 120 (H2BubiK120). *p < 0.05 and ***p = 0.0001.

Article Snippet: The sequence (AGACCC GGGCGGTTGTCCAC) was inserted into the 293-T backbone and then cloned into the CRISPR gRNA vector (#44248 Addgene).

Techniques: CRISPR, Sequencing, Generated, Plasmid Preparation, Control, Transfection, Western Blot, Expressing, Chromatin Immunoprecipitation, Phospho-proteomics

Journal: The Journal of Neuroscience

Article Title: Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation

doi: 10.1523/jneurosci.1453-23.2023

Figure Lengend Snippet: Figure 4. Manipulation of RPT6 leads to a negative correlation with Egr2 expression. A simple linear regression was conducted to identify potential correlations between RPT6 bound at the 3′ UTR of Egr2 (independent variable) and Egr2 expression (dependent variable). To calculate correlation, we collapsed animals across all three experiments (siRNA, CRISPR- dCas9, and CRISPR-dCas13) within each group (control naïve, control trained, or treatment trained). A, There was a trend for a negative correlation in control naïve animals across all experiments. B, No significant correlation was observed in control trained animals across all experiments. C, Trained animals that received a manipulation targeting RPT6 (siRNA knock- down, dCas9-mediated gene placement, or dCas13 targeting of S120) had a significant neg- ative correlation, suggesting that disruption of homeostatic RPT6 levels/activity leads to dysregulated Egr2 expression following training.

Article Snippet: The sequence (AGACCC GGGCGGTTGTCCAC) was inserted into the 293-T backbone and then cloned into the CRISPR gRNA vector (#44248 Addgene).

Techniques: Expressing, CRISPR, Control, Knockdown, Disruption, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation

doi: 10.1523/jneurosci.1453-23.2023

Figure Lengend Snippet: Figure 5. The serine 120 (S120) codon is not necessary for context fear memory formation in the CA1 region of male rats. A, Male rats received a bilateral infusion of either gRNA targeting S120 and dCas13b-ADAR2DD plasmids (RPT6 + Cas13) or dCas13b-ADAR2DD plasmid alone (Cas13 control) into area CA1 of the dorsal hippocampus. After 28 d, rats were trained to standard contextual fear conditioning and then tested 24 h later. B, C, dCas13 targeting of S120 did not impact performance during training (B) nor did it alter memory retention during testing (C). D, Proteasome activity measured in samples collected 1 h after testing was not different between groups for Suc-LLVY chymotrypsin-like, Z-LLE peptidyl-glutamyl or Bz-VGR trypsin-like activity. E, Immunoprecipitation (IP) of RPT6 using CA1 whole-cell extract was completed to confirm knockdown of phosphorylation at serine 120 of RPT6 (pRPT6-S120). F, Representative image of IP of RPT6 from CA1 whole-cell extract of Cas13 control and RPT6 + Cas13 trained animals for quantitative comparison with RPT6 in the top image and pRPT6-S120 in the bottom image. G, The ratio of pRPT6-S120 levels to precipitated RPT6 levels was higher in Cas13 control animals compared with RPT6 + Cas13 animals confirming the knockdown of pRPT6-S120. *p < 0.05 compared to Cas13 control.

Article Snippet: The sequence (AGACCC GGGCGGTTGTCCAC) was inserted into the 293-T backbone and then cloned into the CRISPR gRNA vector (#44248 Addgene).

Techniques: Plasmid Preparation, Control, Activity Assay, Immunoprecipitation, Knockdown, Phospho-proteomics, Comparison