Journal: iScience

Article Title: The noncoding circular RNA circHomer1 regulates synaptic development and experience-dependent plasticity in the mouse visual cortex

doi: 10.1016/j.isci.2025.113421

Figure Lengend Snippet: Experience-dependent circRNAs in V1 identified through MD (A) Timeline and schematic of 3-day MD RNAseq screen. (B) Volcano plot of the mRNA sequencing of V1 after 3-day MD ( n = 3 mice per group). (C) Volcano plot of the circRNA sequencing of V1 after 3-day MD ( n = 3 mice per group). The ipsilateral hemisphere (ipsi) served as a control for MD induced changes in the contralateral hemisphere (contra) for both mRNA and circRNA expression. (D) Fold change of differentially expressed circRNAs and their mRNA isoforms after 3-day MD. The mouse and brain silhouette were adapted from https://doi.org/10.5281/zenodo.8044766 and https://doi.org/10.5281/zenodo.3925971 . The datapoints in green indicate circHomer1 , circPrkce , and circDcun1d4 , whose circular and linear isoforms exhibit a change in the opposite direction after 3-day MD.

Article Snippet: AAV9-U6- circHomer1 shRNA-CMV-DsRed , Vigene , N/A.

Techniques: Sequencing, Control, Expressing

Journal: iScience

Article Title: The noncoding circular RNA circHomer1 regulates synaptic development and experience-dependent plasticity in the mouse visual cortex

doi: 10.1016/j.isci.2025.113421

Figure Lengend Snippet: circHomer1 expression is regulated by experience-dependent plasticity and upregulated during the ocular dominance critical period (A) The mouse Homer1 gene encodes long and short versions of the synaptic protein Homer1 and a noncoding circRNA, circHomer1. (B) RT-qPCR quantification of selected mRNAs and circHomer1 after 3-day (left) or 7-day (right) MD in contralateral V1 relative to ipsilateral V1 ( n = 3–4 mice per group, one sample t-test). (C) circHomer1 (left), Homer1a (middle), and circTulp4 (right) expression measured by RT-qPCR at several timepoints in mouse V1 across development and into adulthood ( n = 3–6 mice per group, one-way ANOVA following Tukey multiple comparisons). Yo, years old. Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: AAV9-U6- circHomer1 shRNA-CMV-DsRed , Vigene , N/A.

Techniques: Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: The noncoding circular RNA circHomer1 regulates synaptic development and experience-dependent plasticity in the mouse visual cortex

doi: 10.1016/j.isci.2025.113421

Figure Lengend Snippet: circHomer1 depletion delays the expression of ocular dominance plasticity in V1 following MD (A) sh-circHomer1 specifically reduces the expression of circHomer1 , but not linear Homer1 , in mouse V1 ( n = 4 mice per group, Student’s t test). (B) sh-circHomer1 has no effect on total Homer1 protein levels as measured by Western blot ( n = 3 mice per group). (C) Mice were injected with either sh-circHomer1 or sh-scramble virus at P15, a craniotomy was performed at P22 to implant a cranial window, then a preMD optical imaging session was done, and the contralateral eye-lid sutured at P25. After either 3-day or 7-day MD, the eyelid was reopened, and post-MD imaging done. (D) Example retinotopic maps from a sh-circHomer1 mouse, obtained with optical imaging. The color corresponds with different phases of the visual stimulus (consistent with the visual field map) and brightness shows the amplitude of the cortical response. Top, contralateral-eye responses, preMD (left) and after 7-day MD (right). Bottom, ipsilateral-eye responses. Scale bar = 500 μm (E) Ocular dominance index (ODI) changes after 3-day or 7-day MD for mice injected with sh-scramble or sh-circHomer1 virus. Each dot shows the average ODI for one animal, and gray lines show the change of ODI for one animal ( n = 5–11 mice per group, mixed-effects model following Holm-Sidak multiple comparisons test). (F) Normalized response amplitudes driven by the contralateral (left) and ipsilateral (right) eyes ( n = 5–11 mice per group, mixed-effects model following Holm-Sidak multiple comparisons test). Data are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: AAV9-U6- circHomer1 shRNA-CMV-DsRed , Vigene , N/A.

Techniques: Expressing, Western Blot, Injection, Virus, Optical Imaging, Imaging

Journal: iScience

Article Title: The noncoding circular RNA circHomer1 regulates synaptic development and experience-dependent plasticity in the mouse visual cortex

doi: 10.1016/j.isci.2025.113421

Figure Lengend Snippet: Depletion of circHomer1 affects spine morphology of V1 neurons (A) A schematic of tissue preparation and eMAP. (B) Neurons expressing both GFP and RFP (denoting shRNA) were selected for analysis (left). Dendritic spines were classified into four categories based on their morphology (right). Scale bars = 100 μm (left, estimated to be 33.33 μm prior to 3× expansion), and 10 μm (right, estimated to be 3.33 μm prior to 3× expansion). H, head width; N, neck width; L, length; H ¯ , average head width in the No MD sh-scramble group. (C) Representative images showing apical dendrites of layer 2/3 neurons in V1 in the No MD, 3-day MD, and 7-day MD condition from the sh-scramble (top) or sh-circHomer1 (bottom) group. Arrows indicate different types of dendritic spines. Scale bar = 10 μm (estimated to be 3.33 μm prior to 3× expansion). (D) Density of mushroom spines, and immature spines (stubby spines, thin spines and filopodia) on apical dendrites of layer 2/3 neurons in V1 in the No MD, 3-day MD, and 7-day MD condition from the sh-scramble (gray) or sh-circHomer1 (pink) group (n = 8–10 dendrites from 3 mice per group, values were normalized to 3× expansion factor, two-way ANOVA following Tukey multiple comparisons). (E) Spine morphology types as a percentage of total spines. The percentage of mushroom spines is labeled on the bar. (F) Volume of dendritic spines on apical dendrites (same data as in (D) and (E), values were normalized to 3 3 expansion factor, two-way ANOVA following Tukey multiple comparisons). (G) Distribution of spine volume in sh-scramble and sh-circHomer1 mice in the 3 conditions ( n = 235–334 dendritic spines from 3 mice per group). Data are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: AAV9-U6- circHomer1 shRNA-CMV-DsRed , Vigene , N/A.

Techniques: Expressing, shRNA, Labeling

Journal: The Journal of Reproduction and Development

Article Title: AVPV Kiss1 neuron-specific knockdown of purinergic P2X2 receptor suppresses LH surge and ovulation in Kiss1-Cre rats

doi: 10.1262/jrd.2024-046

Figure Lengend Snippet: DNA sequences of shRNAs against rat P2rx2

Article Snippet: To examine the effects of AVPV kisspeptin neuron-specific knockdown of P2rx2 mRNA expression on E2-induced LH surge, Kiss1 - Cre female rats were bilaterally injected with AAV-U6- P2rx2 -shRNA-flox-CMV-EGFP (AAV- P2rx2 -shRNA-EGFP, 3.1 × 10 13 viral genome/ml; n = 8) or AAV-U6-scrambled-shRNA-flox-CMV-EGFP (AAV-scrambled-shRNA-EGFP, 2.5 × 10 13 viral genome/ml; n = 4) into the AVPV region through a cannula (C315I, Plastics One, Roanoke, VA, USA) with its tip at 0.12 mm posterior to the bregma, ± 0.5 mm from the midline, and 8.2 mm below the surface of the skull according to the coordinates of a rat brain atlas [ ].

Techniques: Sequencing, shRNA

Journal: The Journal of Reproduction and Development

Article Title: AVPV Kiss1 neuron-specific knockdown of purinergic P2X2 receptor suppresses LH surge and ovulation in Kiss1-Cre rats

doi: 10.1262/jrd.2024-046

Figure Lengend Snippet: Evaluation of the efficacy of siRNA in suppressing P2rx2 mRNA expression in the mHypo-A51 cell line. A: mHypoA-51 cells were transfected with one of three P2rx2 siRNA constructs (siRNA#1, #2, or #3) or negative-control siRNA (siRNA-NC). The cells were incubated for 24 or 48 h and then treated with E2 for 4 h prior to collection of the cells to analyze P2rx2 mRNA levels. P2rx2 mRNA expression levels were measured by real-time RT-PCR. B: P2rx2 mRNA expression levels in mHypo-A51 cells 24 or 48 h after the transfection of P2rx2 siRNA or siRNA-NC. Opti-MEM, non-transfected control. Values are the mean ± SEM. Numbers in each column indicate the number of samples used. Circles on the bar charts indicate the individual data. Different letters indicate statistically significant differences ( p < 0.05; one-way ANOVA followed by the Bonferroni test). C: The target sequence of the rat P2rx2 shRNA corresponding to the mouse P2rx2 target sequence of siRNA#2. The base-mismatches are highlighted in magenta. The sequences are shown from 5' to 3', left to right. siRNA, small interfering RNA; SEM, standard error of the mean; ANOVA, analysis of variance.

Article Snippet: To examine the effects of AVPV kisspeptin neuron-specific knockdown of P2rx2 mRNA expression on E2-induced LH surge, Kiss1 - Cre female rats were bilaterally injected with AAV-U6- P2rx2 -shRNA-flox-CMV-EGFP (AAV- P2rx2 -shRNA-EGFP, 3.1 × 10 13 viral genome/ml; n = 8) or AAV-U6-scrambled-shRNA-flox-CMV-EGFP (AAV-scrambled-shRNA-EGFP, 2.5 × 10 13 viral genome/ml; n = 4) into the AVPV region through a cannula (C315I, Plastics One, Roanoke, VA, USA) with its tip at 0.12 mm posterior to the bregma, ± 0.5 mm from the midline, and 8.2 mm below the surface of the skull according to the coordinates of a rat brain atlas [ ].

Techniques: Expressing, Transfection, Construct, Negative Control, Incubation, Quantitative RT-PCR, Control, Sequencing, shRNA, Small Interfering RNA

Journal: The Journal of Reproduction and Development

Article Title: AVPV Kiss1 neuron-specific knockdown of purinergic P2X2 receptor suppresses LH surge and ovulation in Kiss1-Cre rats

doi: 10.1262/jrd.2024-046

Figure Lengend Snippet: AVPV kisspeptin neuron-specific knockdown of P2rx2 suppressed E2-induced LH surge in OVX Kiss1 - Cre female rats. A: AAV vectors carrying P2rx2 -specific or scrambled shRNA were injected into the bilateral AVPV regions of Kiss1 - Cre female rats. The area outlined by the dotted line indicates the Cre-dependent expression of shRNAs in AVPV kisspeptin neurons of Kiss1 - Cre rats. The AAV vectors were designed to express either P2rx2 -specific shRNA or non-specific scrambled shRNA under the human U6 promoter in a Cre-dependent manner, along with an EGFP reporter under a separate CMV promoter. The shRNA is conditionally expressed in AVPV kisspeptin neurons through Cre-mediated deletion, which excises the stop cassette positioned between the sense (shRNA S.) and antisense (shRNA A.S.) sequences. Fourteen days after the injection of AAV-U6- P2rx2 -shRNA-flox-CMV-EGFP (AAV- P2rx2 -shRNA-EGFP, n = 8) or AAV-U6-scrambled-shRNA-flox-CMV-EGFP (AAV-scrambled-shRNA-EGFP, n = 4), the Kiss1 - Cre rats were ovariectomized (OVX) and implanted with diestrus levels of estradiol-17β (low E2) for 5 days and proestrus levels of E2 (high E2) for 2 days. Blood samples were collected from freely moving rats every 1 h from 1000 h to 2100 h. B: Images of brain sections of representative Kiss1 - Cre rats transfected with AAV-scrambled-shRNA-EGFP or AAV- P2rx2 -shRNA-EGFP showing EGFP expression in the AVPV-preoptic region but not in the ARC. Arrowheads indicate magnified EGFP-expressing cells in the insets. 3V, third ventricle; scale bars, 100 µm. C: Representative images of Kiss1 -expressing (magenta) and EGFP-immmunopositive (green) cells in the AVPV of Kiss1-Cre rats injected with scrambled shRNA (left) or P2rx2 shRNA (right). Insets show magnified images of Kiss1 -expressing EGFP-immunopositive cells indicated by arrowheads. D: The number of Kiss1 -expressing (magenta) and Kiss1 -expressing EGFP-immunopositive (striped) cells in the AVPV of Kiss1-Cre rats injected with scrambled shRNA or P2rx2 shRNA. The number of Kiss1 -expressing cells, as well as the number of Kiss1 -expressing EGFP-immunopositive cells in the AVPV of OVX + high E2 Kiss1-Cre rats in the P2rx2 shRNA group (n = 8) and scrambled shRNA group (n = 4), were not significantly different (P = 0.116 and P = 0.054, respectively; Student’s t -test). E: Plasma LH profiles in OVX + high E2 Kiss1 - Cre rats treated with AVPV administration of P2rx2 shRNA (n = 8, indicated by red circles) or scrambled shRNA (n = 4, indicated by blue circles). Values with * in the P2rx2 -shRNA-treated group were significantly lower than scrambled-shRNA-treated controls at each time point (P < 0.05, Two-way repeated measures ANOVA followed by simple main effects). F: The AUC of plasma LH levels was significantly lower (* P = 0.0129, Student’s t -test) in Kiss1 - Cre rats injected with P2rx2 shRNA compared to the corresponding value of the control Kiss1 - Cre rats injected with scrambled shRNA. Values represent the mean ± SEM. Numbers in each column indicate the number of animals used. Circles on the bar chart indicate the individual data. AVPV, anteroventral periventricular nucleus; LH, luteinizing hormone; AAV, adeno-associated virus; shRNA, short hairpin RNA; EGFP, enhanced green fluorescent protein; ARC, arcuate nucleus; AUC, area under the curve; ANOVA, analysis of variance; SEM: standard error of the mean.

Article Snippet: To examine the effects of AVPV kisspeptin neuron-specific knockdown of P2rx2 mRNA expression on E2-induced LH surge, Kiss1 - Cre female rats were bilaterally injected with AAV-U6- P2rx2 -shRNA-flox-CMV-EGFP (AAV- P2rx2 -shRNA-EGFP, 3.1 × 10 13 viral genome/ml; n = 8) or AAV-U6-scrambled-shRNA-flox-CMV-EGFP (AAV-scrambled-shRNA-EGFP, 2.5 × 10 13 viral genome/ml; n = 4) into the AVPV region through a cannula (C315I, Plastics One, Roanoke, VA, USA) with its tip at 0.12 mm posterior to the bregma, ± 0.5 mm from the midline, and 8.2 mm below the surface of the skull according to the coordinates of a rat brain atlas [ ].

Techniques: Knockdown, shRNA, Injection, Expressing, Transfection, Clinical Proteomics, Control, Virus

Journal: The Journal of Reproduction and Development

Article Title: AVPV Kiss1 neuron-specific knockdown of purinergic P2X2 receptor suppresses LH surge and ovulation in Kiss1-Cre rats

doi: 10.1262/jrd.2024-046

Figure Lengend Snippet: AVPV kisspeptin neuron-specific knockdown of P2rx2 disrupted spontaneous LH surge, ovulation, and estrous cyclicity in ovary-intact Kiss1 - Cre female rats. A: After the AVPV injection of AAV- P2rx2- shRNA-EGFP or AAV-scrambled-shRNA-EGFP, vaginal smears were monitored daily for at least 21 days, and the blood samples were collected to examine plasma LH levels on the day of proestrus or corresponding day and ovulated oocytes were collected on the day after the blood sampling. B: Estrous cyclicity from 8 days before AAV injection until at least 3 weeks after AAV scrambled shRNA or P2rx2 shRNA injection in Kiss1 - Cre rats. Four out of five ovary-intact control Kiss1 - Cre rats showed normal 4-day estrous cycles throughout the experimental period, while three of four ovary-intact AVPV kisspeptin neuron-specific P2rx2 knocked-down Kiss1 - Cre rats showed persistent diestrus. E, estrus; P, proestrus; D, diestrus. Individual plasma LH profiles measured in ovary-intact rats treated with either AVPV scrambled shRNA (C) or P2rx2 shRNA (D). E: The AUC of plasma LH levels in Kiss1-Cre rats that were in proestrus on the day of blood sampling in scrambled shRNA- (animal #1–#4, n = 4) or P2rx2 shRNA-treated Kiss1 - Cre rat (#7, n = 1). F: AVPV administration of P2rx2 shRNA blocked ovulation in the ovary-intact Kiss1 - Cre rat (#7, n = 1), whereas control rats (#1–#4, n = 4) showed ovulation at estrus. G: Images of ovaries in representative Kiss1 - Cre rats with AVPV injection of scrambled shRNA (#4, left) or P2rx2 shRNA (#7, right). Scale bar, 2 mm. Arrowheads, corpora lutea. H: Ovarian weights (total of left and right) of the Kiss1 - Cre rat injected with P2rx2 shRNA (#4, n = 1) and the control rats (#1–#4, n = 4) injected with scrambled shRNA. Values are represented as mean ± SEM. Circles on the bar charts indicate the individual data. AVPV, anteroventral periventricular nucleus; LH, luteinizing hormone; AAV, adeno-associated virus; shRNA, short hairpin RNA; EGFP, enhanced green fluorescent protein; SEM, standard error of the mean.

Article Snippet: To examine the effects of AVPV kisspeptin neuron-specific knockdown of P2rx2 mRNA expression on E2-induced LH surge, Kiss1 - Cre female rats were bilaterally injected with AAV-U6- P2rx2 -shRNA-flox-CMV-EGFP (AAV- P2rx2 -shRNA-EGFP, 3.1 × 10 13 viral genome/ml; n = 8) or AAV-U6-scrambled-shRNA-flox-CMV-EGFP (AAV-scrambled-shRNA-EGFP, 2.5 × 10 13 viral genome/ml; n = 4) into the AVPV region through a cannula (C315I, Plastics One, Roanoke, VA, USA) with its tip at 0.12 mm posterior to the bregma, ± 0.5 mm from the midline, and 8.2 mm below the surface of the skull according to the coordinates of a rat brain atlas [ ].

Techniques: Knockdown, Injection, shRNA, Clinical Proteomics, Sampling, Control, Virus

Journal: Cells

Article Title: AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

doi: 10.3390/cells10020324

Figure Lengend Snippet: Primer Pairs Used for RT-qPCR analysis. Primer sequences (F, forward; R, reverse) and Amplicon Sizes are Based on Species-Specific cDNA Sequences. Amplicon Sizes of gapdh fragments are Indicated for Amplification on cDNA (150 bp) or on Genomic DNA (284 bp), thus allowing to monitor genomic contaminations.

Article Snippet: The sgRNA scaffold, including the hU6 promoter and a dSaCas9-encoding construct, were a gift from Feng Zhang (Addgene (Watertown, MA, USA) plasmid #61591 for the sgRNA scaffold and SaCas9; plasmid #61594 for dSaCas9) [ ].

Techniques: Amplification

Journal: Cells

Article Title: AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

doi: 10.3390/cells10020324

Figure Lengend Snippet: Construction of shRNA-encoding and all-in-one dSaCas9-encoding vectors for Adeno-associated viruses (AAV)‑mediated RNA interference (RNAi) and CRISPRi. ( A a ) Schematic representation of the shRNA expression plasmid. ( A b ) Schematic representation of shRNA-encoding constructs used in this study. Expression of the eGFP reporter could be driven by the CMV- or CKII promoter. ( B a ) Schematic representation of the dSaCas9 and sgRNA expression plasmid. ( B b ) Schematic representation of sgRNA and dSaCas9-encoding constructs used in this study. Expression of the eGFP reporter could be driven by the CMV- or CKII promoter. For control experiments, variants with dSaCas9 fused to eGFP were generated. The modulatory design of the vector also allows to exchange dSaCas9 to SaCas9 for conventional CRISPR experiments. Abbreviations: ITR, inverted terminal repeat; hU6, human U6 promoter; shRNA, short-hairpin RNA; CKII, CaM kinase II promoter; CMV, cytomegalovirus promoter; eGFP, enhanced green fluorescent protein; Ori, origin of replication; Amp r , ampicillin resistance cassette; NLS, nuclear localization site; dSaCas9, nuclease deficient Staphylococcus aureus Cas9; HA, human influenza hemagglutinin tag; KRAB, Krüppel-associated box; sgRNA, short-guidance RNA; Kan r , kanamycin resistance cassette.

Article Snippet: The sgRNA scaffold, including the hU6 promoter and a dSaCas9-encoding construct, were a gift from Feng Zhang (Addgene (Watertown, MA, USA) plasmid #61591 for the sgRNA scaffold and SaCas9; plasmid #61594 for dSaCas9) [ ].

Techniques: shRNA, Expressing, Plasmid Preparation, Construct, Generated, CRISPR

Journal: Cells

Article Title: AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

doi: 10.3390/cells10020324

Figure Lengend Snippet: Expression of RNAi- and Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi)-mediating constructs in HEK293 cells. ( A a – A c ) Representative immunofluorescence images showing the expression of the ( A a ) eGFP reporter of the RNAi construct, ( A b ) dSaCas9 protein including the KRAB domain and the sgRNA expression scaffold, and ( A c ) eGFP-tagged dSaCas9 in HEK293 cells constitutively expressing the hyperpolarization activated and cyclic nucleotide-gated (HCN) channel isoform 1 (HCN1). The eGFP, HA-tag, and HCN1 proteins were immunostained with specific antibodies. ( B a – B c ) Representative immunofluorescence images showing the expression of ( B a ) the eGFP reporter of sh1-expressing, ( B b ) sh2-expressing, or ( B c ) sh4-expressing variants in HEK293 cells constitutively expressing HCN channel isoforms 1, 2, or 4. Staining was performed with specific anti-eGFP, anti-HA-tag, and anti-HCN antibodies combined with fluorescently labeled secondary antibodies (green and red). Nuclei were labeled with TOPRO (blue). ( C a – C c ) Colocalization analysis by comparison of Pearson’s R values for HEK293 cells ( C a ) constitutively expressing HCN1 channels and different shRNAs (shScr, sh1, sh2 and sh4), ( C b ) constitutively expressing HCN2 channels and different shRNAs (shScr, sh1, sh2 and sh4), and ( C c ) constitutively expressing HCN4 channels and different shRNAs (shScr, sh1, sh2 and sh4). The data were obtained from indicated numbers of fluorescent images from at least five independent transfections. Pearson’s R values were normalized to shScr controls and results are depicted as mean ± standard deviation. Schematic of AAV-delivered constructs are displayed above the merged immunofluorescence images.

Article Snippet: The sgRNA scaffold, including the hU6 promoter and a dSaCas9-encoding construct, were a gift from Feng Zhang (Addgene (Watertown, MA, USA) plasmid #61591 for the sgRNA scaffold and SaCas9; plasmid #61594 for dSaCas9) [ ].

Techniques: Expressing, CRISPR, Construct, Immunofluorescence, Staining, Labeling, Transfection, Standard Deviation

Journal: Cells

Article Title: AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

doi: 10.3390/cells10020324

Figure Lengend Snippet: AAV-mediated expression of different constructs in primary hippocampal neurons (PHNs) and organotypic hippocampal slice cultures (OHSCs). ( A ) Schematic representation of the preparation and transduction procedure of primary hippocampal neurons (PHNs). ( B a – B c ) Representative immunofluorescence images of rAAV9-transduced PHNs expressing the ( B a ) eGFP reporter, ( B b ) HA-tagged dSaCas9, and ( B c ) eGFP-tagged dSaCas9. ( C ) Schematic representation of the preparation and transduction procedure for OHSCs. For details see Material and Methods. ( D a – D c ) Representative immunofluorescence images showing rAAV9-transduced OHSCs expressing the eGFP reporter ( D a , D b ), or eGFP-tagged dSaCas9 ( D c ). The eGFP, HA-tag, and the neuron-specific microtubule-associated protein 2 (MAP2) protein were immunostained with specific anti-GFP, anti-HA, and anti-MAP2 antibodies combined with fluorescently labeled secondary antibodies (eGFP and HA-tag, green; MAP2, red). Nuclei were labeled with TOPRO (blue). Schematic of AAV-delivered constructs are displayed above the merged immunofluorescence images.

Article Snippet: The sgRNA scaffold, including the hU6 promoter and a dSaCas9-encoding construct, were a gift from Feng Zhang (Addgene (Watertown, MA, USA) plasmid #61591 for the sgRNA scaffold and SaCas9; plasmid #61594 for dSaCas9) [ ].

Techniques: Expressing, Construct, Transduction, Immunofluorescence, Labeling

Journal: Cells

Article Title: AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

doi: 10.3390/cells10020324

Figure Lengend Snippet: RNAi and CRISPRi reduce the amount of HCN channel transcripts in PHNs and OHSCs. ( A ) qRT-PCR analysis of transcript expression levels for HCN isoforms 1, 2, and 4 in primary hippocampal neurons (PHNs). Transcript expression levels were normalized to gapdh. Values shown are calculated to 1 as the sum of all hcn transcripts. cDNA was prepared from 5 coverslips with PHNs from at least three different animals. ( B a – B c ) Representative immunofluorescence images showing expression of HCN-channel isoforms 1 (green), 2 (blue) and 4 (red). Isoforms were stained using specific antibodies combined with fluorescently labeled secondary antibodies. ( C a ) Schematic representation of constructs delivered by rAAV2 to PHNs. ( C b – C d ) qRT-PCR analysis of hcn1 , hcn2 , and hcn4 mRNA levels in hippocampal neurons after transduction with shRNA or sgRNA/dSaCas9 expressing rAAVs. ( D a ) Schematic representation of constructs delivered by rAAV9 to PHNs. ( D b – D d ) qRT-PCR analysis of hcn1 , hcn2 , and hcn4 mRNA levels in hippocampal neurons after transduction with shRNA or sgRNA/dSaCas9 expressing rAAVs. ( E ) qRT-PCR analysis of transcript expression levels for HCN isoforms 1, 2, and 4 in organotypic hippocampal slice cultures (OHSCs). The transcript expression levels were normalized to gapdh. Values shown are calculated to 1 as the sum of all hcn transcripts. cDNA was prepared from five culture inserts, each containing three individual slices. In total, slices were obtained from three different animals. ( F a , F b ) Representative immunofluorescence images showing the expression of HCN-channel isoforms 1 (green) and 2 (blue). The isoforms were stained using specific antibodies. Enlargements show HCN-isoform expression in hippocampal CA1 (1) and CA3 (2) subfields. ( G a , G b ) qRT-PCR knock-down analysis of hcn2 mRNA levels in organotypic slices after transduction with shRNA or sgRNA/dSaCas9 expressing rAAV9. The data were obtained from indicated numbers of culture inserts, each containing three individual slices. In total, slices were obtained from at least three different animals. The results are depicted as mean ± standard deviation. Statistical significance was assessed using the unpaired two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sgRNA scaffold, including the hU6 promoter and a dSaCas9-encoding construct, were a gift from Feng Zhang (Addgene (Watertown, MA, USA) plasmid #61591 for the sgRNA scaffold and SaCas9; plasmid #61594 for dSaCas9) [ ].

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Labeling, Construct, Transduction, shRNA, Standard Deviation, Two Tailed Test