Journal: bioRxiv

Article Title: Cell type- and stage-specific expression of Otx2 is coordinated by a cohort of transcription factors and multiple cis -regulatory modules in the retina

doi: 10.1101/2019.12.19.882969

Figure Lengend Snippet: (A&B) The O5-sgRNA1 plasmid targets the 5’ end of the O5 CRM in the mouse genome, and the O5-sgRNA2 plasmid targets the 3’ of the O5 CRM. The O5-Cas9 plasmid expressed Cas9 under the control of the O5 CRM. The CAG-mCherry plasmid served as the electroporation efficiency control. These plasmids were co-electroporated into P0 retinas in vivo. At P14, retinas were harvested and dissociated into single cells. The transcript levels of Otx2 in mCherry+Chx10 (Vsx2)+ bipolar cells were detected and quantified by smFISH. (C & D) The level of the Otx2 transcript in control retinal cells. mCherry marked the electroporated cells. Chx10 IHC labeled bipolar cells. The Otx2 transcript was detected by smFISH (Green signal). Each green dot in C4 represents one mRNA molecule. D1-D4 are high magnification views of the highlighted region in C1. White arrows: mCherry+Chx10+ bipolar cells. (E & F) The level of Otx2 transcripts in retinal cells that had the CRISPR constructs to delete the O5 CRM. F1-F4 are high magnification views of the highlighted region in E1. (G) Quantification of the Otx2 transcript levels. The Y axis repre-sents the number of Otx2 transcripts (green dots) in individual bipolar cells based on smFISH (C4, D3, E4 and F3). Solid circles in the plot represents cells with each solid circle representing one cell. The percentage of cells that expressed different levels of Otx2 are indicated. **: P<0.01. Scale bar: 10um.

Article Snippet: The Chx-mCherry-lacZ-shRNA or Chx-mCherry-Otx2-shRNA plasmid was generated by replacing the CAG promoter of the CAG-mCherry-lacZ-shRNA (Addgene #73978, ( )) or CAG-mCherry-Otx2-shRNA (Addgene #73980, ) plasmid with the 164bp Chx10 CRM and basic TATA box promoter ( ).

Techniques: Plasmid Preparation, Electroporation, In Vivo, Labeling, CRISPR, Construct

Journal:

Article Title: Serum-Induced Expression of the cdc25A Gene by Relief of E2F-Mediated Repression

doi:

Figure Lengend Snippet: Serum induction of cdc25A promoter deletions. The constructs indicated at the bottom of the figure were transfected into NIH 3T3 cells with pRL-SV40P as an internal control and analyzed for serum induction as described in the legend to Fig. ​Fig.2A.2A. A minimal c-fos promoter plasmid, p0-Fluc (see Fig. ​Fig.5),5), was tested as a control. Shown are the averages of data from three experiments and the standard errors of the means (error bars). − serum, no serum induction; + serum, serum induction performed.

Article Snippet: The internal control plasmid pRL-SV40P was generated by placing the simian virus 40 (SV40) promoter from pGL3-Promoter (Promega) into pRL-TK (Promega) such that the SV40 promoter was driving the Renilla luciferase gene.

Techniques: Construct, Transfection, Plasmid Preparation

Journal:

Article Title: Serum-Induced Expression of the cdc25A Gene by Relief of E2F-Mediated Repression

doi:

Figure Lengend Snippet: Serum induction of cdc25A promoter constructs. (A) NIH 3T3 cells were transfected with the indicated cdc25A promoter-luciferase constructs containing nucleotides −450 to +126 of the cdc25A promoter, pNPGL3, and additional genomic fragments of the cdc25A gene (17). An internal control plasmid, pRL-SV40P, expressing Renilla luciferase was cotransfected. The transfected cells were serum starved (−serum) and then induced with 20% serum for 13 h (+ serum). Firefly luciferase activities were measured and normalized to the internal control Renilla luciferase activity. Shown are the averages of data from three experiments and the standard errors of the means (SEM; error bars). (B) NIH 3T3 cells were transfected with the indicated reporter genes and pRL-SV40P, serum starved, and then induced with serum for the indicated periods. The results are the average normalized luciferase activities of three experiments ± the standard errors of the means. NP0.7WT = pNP0.7GL3; NP0.7MUT contains a double point mutation in the myc binding site in NP0.7WT.

Article Snippet: The internal control plasmid pRL-SV40P was generated by placing the simian virus 40 (SV40) promoter from pGL3-Promoter (Promega) into pRL-TK (Promega) such that the SV40 promoter was driving the Renilla luciferase gene.

Techniques: Construct, Transfection, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Mutagenesis, Binding Assay

Journal:

Article Title: Serum-Induced Expression of the cdc25A Gene by Relief of E2F-Mediated Repression

doi:

Figure Lengend Snippet: Serum induction of heterologous-promoter constructs. Regions of the cdc25A promoter were fused upstream of a c-fos minimal promoter construct as indicated at the bottom of the figure (LUC, luciferase gene). The constructs were transfected into NIH 3T3 cells with pRL-SV40P and either not serum induced (− serum) or serum induced (+ serum) as described in the legend to Fig. ​Fig.2A.2A. Shown are the averages of data from three experiments and the standard errors of the means (error bars). The c-fos minimal promoter alone, p0-Fluc, was not inducible (Fig. ​(Fig.44).

Article Snippet: The internal control plasmid pRL-SV40P was generated by placing the simian virus 40 (SV40) promoter from pGL3-Promoter (Promega) into pRL-TK (Promega) such that the SV40 promoter was driving the Renilla luciferase gene.

Techniques: Construct, Luciferase, Transfection

Journal:

Article Title: Serum-Induced Expression of the cdc25A Gene by Relief of E2F-Mediated Repression

doi:

Figure Lengend Snippet: E2F regulation of the cdc25A promoter. (A) The cdc25A E2F site at position −62 was mutated as diagrammed in Fig. ​Fig.44 (pNP-E2F−) and tested for serum induction in NIH 3T3 cells. (B) The cdc25A reporter construct pNPGL3 was transfected with an E2F-1 expression vector or vector pcDNA3 and tested for serum-induced expression. (C) Adenovirus E1A expression vectors were transfected with pNPGL3 and pRL-SV40P. Wild-type E1A (E1A.WT) and mutants were transfected. A plasmid with a frameshift of E1A, pSFS, which does not express E1A served as a control vector. The E1A.RG2 mutant binds Rb family proteins but not p300 or CBP (57). The E1A YH47/928 mutant (E1A.YH) binds p300 and CBP but not Rb family proteins (57). Shown are the averages of data from three experiments and the standard errors of the means (error bars). − serum, no serum induction; + serum, serum induction performed.

Article Snippet: The internal control plasmid pRL-SV40P was generated by placing the simian virus 40 (SV40) promoter from pGL3-Promoter (Promega) into pRL-TK (Promega) such that the SV40 promoter was driving the Renilla luciferase gene.

Techniques: Construct, Transfection, Expressing, Plasmid Preparation, Mutagenesis

Journal: Cell reports

Article Title: Fast Retrograde Access to Projection Neuron Circuits Underlying Vocal Learning in Songbirds

doi: 10.1016/j.celrep.2020.108364

Figure Lengend Snippet: (A) Illustration of the AAV capsid, showing exposed surface proteins responsible for binding to the heparin sulfate proteoglycans (HSPG) receptor, thought to be important for cellular entry of the viral vector. Mutations of two crucial arginines in the heparin binding domain (HBD; highlighted in green) cap sequence at positions 585 and 588 (highlighted in red) reduce heparin binding affinity and potentially improve retrograde transport. Changes are shown for four serotypes that have been used for or possess potential for retrograde transduction. (B) AAV vector genomes encoding diverse transgenes (orange region) including fluorescent proteins and the calcium indicator GCaMP6f. Numbers on top indicate the base pair length and relative position of encoded elements. Note that the actual size of the region encoding the transgene (orange) varies depending on the respective protein. Brainbow stands for three separate AAV vector genomes encoding either eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), or mRuby3 (a red fluorescent protein). (C, F, H, and L) Schematics of the virus injection sites (green) and the neuronal projections analyzed (red). (D and G) Native fluorescence signal in retrogradely labeled HVC X neurons 7 days post-delivery of virus in zebra finch and in canary, respectively.(E, I–K, and M–O) Higher magnification z stack of the boxed region in (D). Dopaminergic projection neurons in zebra finch (I–K) and mouse (M–O) retrogradely labeled with our eGFP-construct, after immunostaining for anti-GFP (I and M) and anti-tyrosine hydroxylase (J and N). Scale bars: 100 μm in (D), (G), (I)–(K), and (M)–(O) and 10 μm in (E).

Article Snippet: For construction of the mRuby3 vector plasmid (pscAAV-2-hCMV-chI-mRuby3-SV40p(A)), the mRuby3 ORF was amplified by PCR using Addgene #85146 as the template DNA and primers 5′-CATTACTAGTGTTTAAACACTCGAGGCTAGCGCCACCATGGTGTCTAAGG-3′ (forward) and 5′-TAGGCGCGCCTACGTACAATTGGGTACCTTACTTGTACAGCTCGTCCATG-3′ (reverse).

Techniques: Binding Assay, Plasmid Preparation, Sequencing, Transduction, Virus, Injection, Fluorescence, Labeling, Construct, Immunostaining

Journal: Cell reports

Article Title: Fast Retrograde Access to Projection Neuron Circuits Underlying Vocal Learning in Songbirds

doi: 10.1016/j.celrep.2020.108364

Figure Lengend Snippet: (A) AAV-mediated brainbow labeling is achieved by injecting a mixture of three individual vectors encoding either eGFP, eCFP, or mRuby3. Each neuron ends up displaying a distinct color because of variable transduction of the three fluorescent proteins, largely depending on the number of vectors per cell per fluorophore. (B) Brainbow labeling of locally transduced area X neurons allows for identification of individual neurons in a densely labeled z stack. (C) High-magnification confocal images reveal consistent color labeling in dendritic fragments of area X neurons, including spines. (D) Retrograde brainbow labeling in entorhinal cortex of neurons projecting to hippocampus in mouse. Scale bars: 100 μm in (B), 20 μm in (C), and 50 μm in (D).

Article Snippet: For construction of the mRuby3 vector plasmid (pscAAV-2-hCMV-chI-mRuby3-SV40p(A)), the mRuby3 ORF was amplified by PCR using Addgene #85146 as the template DNA and primers 5′-CATTACTAGTGTTTAAACACTCGAGGCTAGCGCCACCATGGTGTCTAAGG-3′ (forward) and 5′-TAGGCGCGCCTACGTACAATTGGGTACCTTACTTGTACAGCTCGTCCATG-3′ (reverse).

Techniques: Labeling, Transduction

Journal: Cell reports

Article Title: Fast Retrograde Access to Projection Neuron Circuits Underlying Vocal Learning in Songbirds

doi: 10.1016/j.celrep.2020.108364

Figure Lengend Snippet:

Article Snippet: For construction of the mRuby3 vector plasmid (pscAAV-2-hCMV-chI-mRuby3-SV40p(A)), the mRuby3 ORF was amplified by PCR using Addgene #85146 as the template DNA and primers 5′-CATTACTAGTGTTTAAACACTCGAGGCTAGCGCCACCATGGTGTCTAAGG-3′ (forward) and 5′-TAGGCGCGCCTACGTACAATTGGGTACCTTACTTGTACAGCTCGTCCATG-3′ (reverse).

Techniques: Virus, Recombinant, Plasmid Preparation, Software