circular pcmv6-ac-gfp plasmid containing the coding sequence for pcsk9  (OriGene)

Journal: International Journal of Molecular Sciences

Article Title: The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 ( PCSK9 ) Gene Expression in a Cell-Free Transcription/Translation System

doi: 10.3390/ijms25031463

Figure Lengend Snippet: Schematic graph presenting the crucial role of proprotein convertase subtilisin/kexin type 9 (PCSK9) on LDL receptor turnover in hepatocytes (created with BioRender.com , accessed on 15 November 2023).

Article Snippet: A circular pCMV6-AC-GFP plasmid containing the coding sequence for PCSK9 (OriGene Technologies, Rockville, MD, USA, Cat No. RG220000) served as a template.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 ( PCSK9 ) Gene Expression in a Cell-Free Transcription/Translation System

doi: 10.3390/ijms25031463

Figure Lengend Snippet: Integrated optical density (IOD) of DNA bands after conventional reverse transcription PCR (RT–PCR) ( B ) and agarose gel electrophoresis ( A ). An equal amount of RNA taken from the T7 RNA polymerase reaction served as a template. The control sample contained a PNA oligomer that was not complementary to the PCSK9 coding sequence. This sample was arbitrarily assigned a value of 100%. In Panel ( B ), dashed line bars represent real-time PCR, while white dotted black bars represent conventional PCR after 15 cycles, in comparison to the control PNA. *— p < 0.05 vs. ctrlPNA for RT-PCR, #— p < 0.05 vs. ctrlPNA for conventional PCR.

Article Snippet: A circular pCMV6-AC-GFP plasmid containing the coding sequence for PCSK9 (OriGene Technologies, Rockville, MD, USA, Cat No. RG220000) served as a template.

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control, Sequencing, Real-time Polymerase Chain Reaction, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 ( PCSK9 ) Gene Expression in a Cell-Free Transcription/Translation System

doi: 10.3390/ijms25031463

Figure Lengend Snippet: Western blot image of an in vitro transcription/translation reaction product resolved by electrophoresis with appropriate integrated optical density (I.O.D) values ( A ) and the relative amount of protein with respect to control sample ( B ). 1–4: Samples that contained InitPNA, Ex1PNA, Ex2PNA, and control (non-PCSK9 specific) PNA, respectively. M: Color Prestained Protein Standard, Broad Range (New England Biolabs, no. Cat. P7712). The PCSK9-specific band is depicted with an arrow. A total amount of 10 µg (25 µL sample volume) of purified products from the transcription/translation reaction was loaded onto the gel. The initial protein concentration in the samples was assessed spectrophotometrically. Measurements were made using ImageJ 1.53t software [ , ] *: p < 0.05 vs. control.

Article Snippet: A circular pCMV6-AC-GFP plasmid containing the coding sequence for PCSK9 (OriGene Technologies, Rockville, MD, USA, Cat No. RG220000) served as a template.

Techniques: Western Blot, In Vitro, Electrophoresis, Control, Purification, Protein Concentration, Software

Journal: International Journal of Molecular Sciences

Article Title: The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 ( PCSK9 ) Gene Expression in a Cell-Free Transcription/Translation System

doi: 10.3390/ijms25031463

Figure Lengend Snippet: The representative HPLC traces of GFP-tagged PCSK9 synthetic protein after addition of PNA oligomers ( A ). Area under the curve (AUC) values were calculated for each sample (run in triplicates) from chromatography peaks of GFP-tagged PCSK9 protein synthesized with the presence of different PNA oligomers ( B ) *: p < 0.05 vs. CtrlPNA.

Article Snippet: A circular pCMV6-AC-GFP plasmid containing the coding sequence for PCSK9 (OriGene Technologies, Rockville, MD, USA, Cat No. RG220000) served as a template.

Techniques: Chromatography, Synthesized

Journal: International Journal of Molecular Sciences

Article Title: The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 ( PCSK9 ) Gene Expression in a Cell-Free Transcription/Translation System

doi: 10.3390/ijms25031463

Figure Lengend Snippet: Sequences of synthesized PNA-Ahx-NLS conjugates (Ahx-6-aminohexanoic acid).

Article Snippet: A circular pCMV6-AC-GFP plasmid containing the coding sequence for PCSK9 (OriGene Technologies, Rockville, MD, USA, Cat No. RG220000) served as a template.

Techniques: Synthesized, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 ( PCSK9 ) Gene Expression in a Cell-Free Transcription/Translation System

doi: 10.3390/ijms25031463

Figure Lengend Snippet: Properties of PNA oligomers used in this study.

Article Snippet: A circular pCMV6-AC-GFP plasmid containing the coding sequence for PCSK9 (OriGene Technologies, Rockville, MD, USA, Cat No. RG220000) served as a template.

Techniques: Sequencing, Control

Journal: International Journal of Molecular Sciences

Article Title: The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 ( PCSK9 ) Gene Expression in a Cell-Free Transcription/Translation System

doi: 10.3390/ijms25031463

Figure Lengend Snippet: High-performance liquid chromatography (HPLC) conditions for analysis of GFP-tagged PCSK9 protein after cell-free synthesis in vitro. The reaction was carried out under the following conditions: flow: 0.8 mL/min; detection (ex/em): 482/511 nm; column temperature: 40 °C; injection volume: 10 µL; mobile phase A: 0.1% trifluoroacetic acid in deionized water; mobile phase B: 0.1% trifluoroacetic acid in acetonitrile.

Article Snippet: A circular pCMV6-AC-GFP plasmid containing the coding sequence for PCSK9 (OriGene Technologies, Rockville, MD, USA, Cat No. RG220000) served as a template.

Techniques: High Performance Liquid Chromatography, In Vitro, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: PCSK9 deficiency alters brain lipid composition without affecting brain development and function

doi: 10.3389/fnmol.2022.1084633

Figure Lengend Snippet: PCSK9 targets LDLR in neuronal cell cultures but not in adult cerebellum. (A) Schematic representation of the experiment with PCSK9 treatment on primary neuronal cultures. (B) Schematic representation of the assessment of the LDLR levels in the PCSK9 KO mouse cerebellum using ELISA. Representative Western blots of LDLR receptor in cortical (C) and cerebellar granule neurons (D) . Primary cultures were treated with 10 nM or 100 nM concentration of PCSK9 at 1 day in vitro (DIV) and lysed at 3 DIV. Liver samples of WT and PCSK9 KO mice were added as control (C) . Densitometric quantification of LDLR normalized to β-actin in cortical ( n = 6; E ), and cerebellar granule neurons ( n = 4; F ). ELISA of LDLR in cerebellum ( n = 7 WT, 8 KO) of adult mice (3- to 12-month-old; G ). Data are represented as mean ± SEM. Statistical significance was evaluated using a two-tailed Student’s t-test, alpha = 0.05. Asterisks indicate significantly different values (NS p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: For IUE, the PCSK9 coding sequence was derived from the Origene clone RC2200000, the C-terminal FLAG-tag was eliminated and a NotI/EcoRI was fragment was ligated into the pCAGEN vector (Addgene #11160) and sequenced.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Concentration Assay, In Vitro, Control, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: PCSK9 deficiency alters brain lipid composition without affecting brain development and function

doi: 10.3389/fnmol.2022.1084633

Figure Lengend Snippet: Lack of PCSK9 during development does not affect the fate of cortical progenitor cells. (A) Schematic representation of the experiment. (B) Representative images of immunostaining signals (Satb2, Ctip2, and DRAQ5) in WT and PCSK9 KO E17.5 cortices. (C) Quantification of the fraction of all neurons (DRAQ5 labelled) expressing indicated fate markers (Satb2, Ctip2) in WT and PCSK9 KO E17.5 cortical slices (see also ). (D,E) Quantification of laminar distribution of cortical neurons labelled with Satb2 (D) or Ctip2 (E) fate markers in WT and PCSK9 KO E17.5 cortical slices (see also ). Cortical bin – the maximum height of the cortical slice was divided into 5 bins of identical dimensions. Results on graphs are represented as average percent ± SEM. For statistical analyses two-way ANOVA with Bonferroni multiple comparison test was performed, alpha = 0.05. All p values were above 0.05 and considered non-significant; n = 3 WT, 3 KO.

Article Snippet: For IUE, the PCSK9 coding sequence was derived from the Origene clone RC2200000, the C-terminal FLAG-tag was eliminated and a NotI/EcoRI was fragment was ligated into the pCAGEN vector (Addgene #11160) and sequenced.

Techniques: Immunostaining, Expressing, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: PCSK9 deficiency alters brain lipid composition without affecting brain development and function

doi: 10.3389/fnmol.2022.1084633

Figure Lengend Snippet: The effect of PCSK9 deficiency on lipids in cerebellum and cortex. Lipid profiles of mouse cerebellum and cortex were analyzed using a quantitative mass-spectrometry based shotgun lipidomics method. (A) Schematic representation of the experiment. (B,C) Principal component analysis showing different clustering of WT and KO cerebellar (B) and cortical (C) lipidomes. (D) Cholesterol (FC) content in mol% in WT and (Continued)FIGURE 3 (Continued)PCSK9 KO cerebellum and cortex. Data are represented as mean ± SEM. (E) Heatmap of different lipid classes in PCSK9 KO cortex and cerebellum in mol % compared to WT, shown as log2 fold changes. (F–I) Volcano plots representing the significantly up- and down-regulated lipid classes and species in cerebellum (F,G) and cortex (H, I) . N = 9 per group; 13-to 15-week-old male mice. Only statistically significant results showed. Statistical significance was assessed using unpaired two-tailed Student’s t test, alpha = 0.05. See also , for descriptive statistics and individual p values. Asterisks indicate significantly different values (* p ≤ 0.05; ** p ≤ 0.01). Dash indicates the absence the lipid class in the indicated brain area. The variations of LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, and PS with O-in the end are ether lipids.

Article Snippet: For IUE, the PCSK9 coding sequence was derived from the Origene clone RC2200000, the C-terminal FLAG-tag was eliminated and a NotI/EcoRI was fragment was ligated into the pCAGEN vector (Addgene #11160) and sequenced.

Techniques: Mass Spectrometry, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: PCSK9 deficiency alters brain lipid composition without affecting brain development and function

doi: 10.3389/fnmol.2022.1084633

Figure Lengend Snippet: The effect of PCSK9 deficiency on specific lipid species in cerebellum and cortex. Lipid profiles of mouse cerebellum and cortex were studied using a quantitative mass-spectrometry based shotgun lipidomics method. Heatmap of lipid species in PCSK9 KO cortex and cerebellum in mol % compared to WT, shown as log2 fold changes. N = 9 per group; 13-to 15-week-old male mice. Only statistically significant results showed. Statistical significance was assessed using unpaired two-tailed Student’s t test, alpha = 0.05. See also for descriptive statistics and individual p values. Asterisks indicate significantly different values (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). Dash indicates the absence of the lipid species in the brain area.

Article Snippet: For IUE, the PCSK9 coding sequence was derived from the Origene clone RC2200000, the C-terminal FLAG-tag was eliminated and a NotI/EcoRI was fragment was ligated into the pCAGEN vector (Addgene #11160) and sequenced.

Techniques: Mass Spectrometry, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: PCSK9 deficiency alters brain lipid composition without affecting brain development and function

doi: 10.3389/fnmol.2022.1084633

Figure Lengend Snippet: Lipid classes detected in WT and KO cerebellum.

Article Snippet: For IUE, the PCSK9 coding sequence was derived from the Origene clone RC2200000, the C-terminal FLAG-tag was eliminated and a NotI/EcoRI was fragment was ligated into the pCAGEN vector (Addgene #11160) and sequenced.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: PCSK9 deficiency alters brain lipid composition without affecting brain development and function

doi: 10.3389/fnmol.2022.1084633

Figure Lengend Snippet: Lipid classes detected in WT and KO cortex.

Article Snippet: For IUE, the PCSK9 coding sequence was derived from the Origene clone RC2200000, the C-terminal FLAG-tag was eliminated and a NotI/EcoRI was fragment was ligated into the pCAGEN vector (Addgene #11160) and sequenced.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: PCSK9 deficiency alters brain lipid composition without affecting brain development and function

doi: 10.3389/fnmol.2022.1084633

Figure Lengend Snippet: Behavioral characterization of PCSK9 KO mice. (A) Schematic representation of the experiment. (B–D) Barnes maze. Time to reach target (B) . Mean distance to target (C) . Representative examples of search patterns at Day 1 and Day 5 (D) ( N = 11 WT and 8 KO mice per group). (E) Rotarod.(Continued)FIGURE 5 (Continued)Time spent on the rod ( n = 9 WT and 10 KO mice per group). (F–I) Open field test. Distance travelled (F) . Entries to the center zone (G) . Time spent in the center zone (H) . Distance travelled in the center zone (I) ( N = 11 WT and 8 KO mice per group). (J–M) Elevated plus maze. Distance travelled (J) . Open arm entries (K) . Time spent in open arms (L) . Distance travelled in open arms (M) ( N = 11 WT and 8 KO mice per group). (N) Marble burying test. Number of buried marbles ( n = 9 WT and 10 KO mice per group). For all behavioral tests, 9-to-11-week-old male mice were used. All data are represented as mean ± SEM. Statistical significance was evaluated using a two-tailed Student’s t -test ( C , F–N ). Data of panels B,E are analyzed using two-way ANOVA RM. (B) Interaction F (4,68) = 0.4; p > 0.8; Main effect time. (E) Interaction F (3, 51) = 0.20; p > 0.89. Main effect time. Sidak’s multiple comparisons adjusted p values WT §, KO #. Asterisks indicate significantly different values (NS p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: For IUE, the PCSK9 coding sequence was derived from the Origene clone RC2200000, the C-terminal FLAG-tag was eliminated and a NotI/EcoRI was fragment was ligated into the pCAGEN vector (Addgene #11160) and sequenced.

Techniques: Two Tailed Test