Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Eight out of ten constructs successfully modified for expression-pattern characterization in mice

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Construct, Modification, Expressing

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Eight novel Hprt targeted embryonic stem cell lines successfully generated

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Clone Assay, Isolation

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Summary of expression pattern from reporter mouse strains

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Expressing

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Human bacterial artificial chromosomes can be targeted at Hprt by homologous recombination and, if desired, conditionally removed using cre recombinase. (a) Integration into the mouse genome of the bacterial artificial chromosome (BAC)- lacZ -reporter constructs by homologous recombination results in the human gene in either direction relative to the X chromosome; this schematic presents one possible orientation. Regardless of orientation, each insertion resulted in the presence of four loxP sites in the genome (two wild-type and one 511 mutant at one end and one wild-type at the other end of the BAC insert). (b) Crossing the BAC- lacZ- reporter females to ACTB-cre males should result in the generation of two different male offspring; BAC- lacZ -reporter animals, wild-type for the ACTB-cre transgene; and BAC- lacZ- reporter animals carrying the ACTB-cre transgene. Only the reporter animals that are positive for the ACTB-cre gene should recombine the outer most loxP sites, resulting in excision of the BAC construct from the genome and leaving one loxP site. This would result in an absence of lacZ- positive signal. hP , human HPRT promoter; h1, human first exon; m2 and m3, mouse second and third exons; mouse homology arms (dark blue); Hprt coding regions (red); vector backbone (yellow with black edges); Sac B gene from BAC vector backbone (brown); 5′ and 3′ untranslated regions of the human gene (orange); coding region of the human gene (green); lacZ reporter gene (light blue). Schematic, not to scale. (c-f) lacZ expression results from AMOTL1 - lacZ , MAOA - lacZ , NOV - lacZ , and NR2F2 - lacZ females bred to the ACTB-cre males are presented. lacZ -positive staining (blue) was detected in AMOTL1 - lacZ , MAOA - lacZ , NOV - lacZ , and NR2F2 - lacZ males not carrying the ACTB-cre allele whereas absence of staining was detected in males positive for ACTB-cre by genotyping ( AMOTL1 - lacZ , ACTB-cre; MAOA - lacZ , ACTB-cre; NOV - lacZ , ACTB-cre; NR2F2 - lacZ , ACTB-cre), suggesting whole BAC excision from the genome. Scale bar: (c-f) 1 mm. N = 3 animals for all genotypes.

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Homologous Recombination, Construct, Mutagenesis, Plasmid Preparation, Expressing, Staining

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Human NR2F2 - lacZ expressed in mature neurons populating the basolateral and corticolateral amygdaloid nuclei that are immunoreactive for the Nr2f2 mouse protein. Expression analysis of the human NR2F2 - lacZ strain was undertaken by examination of β-galactosidase (β-gal) staining (blue). (a) E12.5 whole embryos revealed staining in the rostral secondary prosencephalon (black arrow) that extended throughout all three prosomeric regions of the diencephalon (black arrowhead). Staining was present in the nasal cavity (white arrow), the vestibulochochlear ganglion (red arrowhead) and mesenchyme of the posterior limbs (white arrowhead). (b) E12.5 cleared embryos additionally demonstrated staining in the developing bladder (black arrow). (c) P7 brains stained in the amygdala nuclei (white arrows), and the subthalamic nuclei (black arrows). (d) Adult brains revealed strong staining extending from the posterior basolateral amygdaloid nuclei (BLP) (black arrows) to the posterolateral cortical amygdaloid nuclei (PLCo) (white arrows), and the posteroventral part of the medial amygdaloid nuclei (MePV) (white arrowheads). Broad staining was detected in the ventral thalamic area, excluding the cerebral peduncle (cp). (e) Colocalization experiment using β-gal staining and a neuronal nuclei (NeuN) antibody (brown) performed on adult brain cryosections revealed strong expression of NR2F2 - lacZ in mature neurons populating the BLP, and the basomedial amygdaloid nuclei (BMP) (red arrows). Colocalization was found in the PLCo, and the posteromedial cortical amygdaloid nuclei (PMCo) (black arrows), and the MePV (black arrowhead). Lower level of β-gal staining was detected in mature neurons in the anterolateral amygdalohippocampal area (AHiAL) (red arrowhead). Boxed region in (e) is shown in (f) . (f) Higher magnification revealed strong expression of β-gal in mature neurons in the PMCo and sparse expression in mature neurons in the AHiAL. (g) Colocalization experiment, using an anti-β-gal antibody (green), and an NR2F2 antibody (red), performed on adult brain cryosections revealed strong β-gal labeling in cells expressing the Nr2f2 mouse gene in brain regions extending from the PMCo (white arrowhead) to the MePV (white arrow). Lower levels of β-gal were detected in the AHiAL (red arrow). Boxed region in (g) is shown in (h) . (h) Higher magnification revealed strong expression of β-gal (green) in Nr2f2 -positive cells (red) in the PMCo (white arrow) and lower expression in the AHiAL (white arrowhead). LV, lateral ventricle. Scale bar: (a - d) 1 mm; (e , g) 100 μm; (f , h) 20 μm.

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Expressing, Staining, Labeling

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Comparative genomics delineated the DNA boundaries that were sufficient for adult brain-specific expression of NOV and NR2F2 . Coordinates corresponding to the human bacterial artificial chromosome (BAC) constructs used in this study were retrieved and visualized using the University of California Santa Cruz (UCSC) genome browser. (a) DNA alignment of the human NOV BAC (RP11-840I14) (black), against both the RP23-235B13 BAC construct used in the Gene Expression Nervous System Atlas (GENSAT) mouse model (blue), and the mouse genome, delineated the genomic DNA boundaries sufficient for proper expression of this human gene in the basolateral amygdaloid nuclei, cortical layers, and pyramidal neurons in the cornu ammonis 1 (CA1) regions in the adult brain. One hypothesis suggested by our results was that additional functionally conserved regulatory elements homologous to the large non-overlapping 3′ mouse-BAC region are necessary for proper human-gene expression in the developing cortical layers at P7. (b) DNA alignment of the human NR2F2 BAC (RP11-134D15) (black), against both the RP23-109L9 BAC construct used in the GENSAT mouse model (blue) and the mouse genome, delineated the genomic DNA boundaries sufficient for region-specific expression of this human gene in the basolateral, and corticolateral amygdaloid nuclei in the adult brain. One hypothesis suggested by our results was that additional functionally-conserved regulatory elements homologous to the non-overlapping 5′ mouse-BAC region are necessary for proper expression in the developing hypothalamus at P7. Black rectangle box in (b) is shown in (c) . (c) Sequence alignment using the coordinates of the primers used in the BAC lacZ retrofitting process (grey bars) and the cDNA sequence used to generate an anti-NR2F2 antibody (black bar), suggested that the absence of expression of the NR2F2 - lacZ constructs in retinal amacrine cells was not attributable to detection of different isoforms of NR2F2 .

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Expressing, Construct, Gene Expression, Sequencing

Journal: BMC Biology

Article Title: Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

doi: 10.1186/1741-7007-11-106

Figure Lengend Snippet: Primers used for reporter-gene retrofitting

Article Snippet: Colocalization experiments were performed using chicken anti-β-gal antibody (Abcam, San Francisco, CA, USA; ab9361) 1:5,000, mouse anti-NR2F2 antibody (R&D systems, Minneapolis, MN, USA; PP-H7147-00) 1:100, and incubated overnight at 4°C.

Techniques: Sequencing

Journal: Nature communications

Article Title: Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis.

doi: 10.1038/s41467-018-07333-4

Figure Lengend Snippet: Fig. 2 Single-cell RNA-seq identified subpopulations of cardiomyocytes. a Late stage cardiomyocytes (4,689 cells with TNNT2 and ACTC1 expression from day 14 and day 45) were further resolved using ICGS to identify subpopulations of cardiomyocytes. Associated t-SNE cell populations were colored by day of differentiation, as well as by cluster identified (markers within each cluster beside each cluster). b Six cardiomyocyte populations were identified representing subpopulations of cardiomyocytes in early proliferative stages (Cluster 4, 5) expressing cyclins, mid-cardiomyocyte stage (Cluster 2, 3) expressing HAND1, and more mature cardiomyocytes (Cluster 0, 1) expressing sarcomeric (MYH6, MYL2, TNNT2, MYBPC3) and calcium handling genes (RYR2, PLN). c Single cells from day 30 of differentiation were profiled using an independent technology (Fluidigm C1) to resolve coincident mid-to-late state differentiation heterogeneity. Genes associated with distinct observed cell populations identified by the algorithm MarkerFinder are shown. Factors with defining expression in each of the HOPACH clustered populations are shown (SF1, ISL1, JARID2, TBX5, MYL2/HEY2, and HOPX). Two major groups in hiPSC-CMs were observed to express either NR2F2 or MYL2/HEY2. Within each group, identified heart developmental regulators were identified using the software GO-Elite (left panel)

Article Snippet: To overexpress NR2F2, TBX5, and HEY2 in hiPSC-CMs, lentiviral particles were purchased for mGFP-tagged NR2F2 (Cat# RC206753L2V), TBX5 (Cat# RC216520L2V), HEY2 (Cat# RC202544L2V), and control GFP Lent-ORF particles (Cat# PS100071V5) from Origene.

Techniques: RNA Sequencing, Expressing, Software

Journal: Genes

Article Title: Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

doi: 10.3390/genes11020120

Figure Lengend Snippet: Effects of NR2F2 knockdown on progesterone biosynthesis. ( A ) gene expression of NR2F2 after siRNA transfection; ( B ) Effects of NR2F2 knockdown on progesterone secretion; ( C ) gene expression of key factors (StAR, Cyp11a1, HSD3B, and NPC1) involved in progesterone biosynthesis; ( D , E ) protein expression of NR5A2, StAR, Cyp11a1, and HSD3B. Primary pGCs were transfected with NR2F2 siRNA (siNR2F2) or scramble siRNA (siNC). Six hours later, the medium was changed to fresh medium with DMSO or DAPT, followed by further culture for 48 h. * means significant difference at p < 0.05.

Article Snippet: The proteins were subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany) and probed with primary antibodies against StAR (DF6192, Affinity Biosciences, Changzhou, China), Cyp11a1 (DF6569, Affinity Biosciences), HSD3B (DF6639, Affinity Biosciences), ERK1/2 (4695T, Cell Signaling Technology, Danvers, MA, USA), pERK1/2 (4370T, Cell Signaling), NR2F2 (NBP1-31980, Novus Biologicals, CO, USA), NR5A2 (NBP2-27196SS, Novus) and β-actin (20536-1-AP, Protein Tech, Wuhan, China).

Techniques: Knockdown, Gene Expression, Transfection, Expressing

Journal: Genes

Article Title: Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

doi: 10.3390/genes11020120

Figure Lengend Snippet: Effects of NR5A2 and NR2F2 knockdown on the expression of Notch receptors and ligands. ( A ) Effects of NR5A2 and NR2F2 on the expression of Notch receptors (Notch2 and Notch3) and Notch ligands (Dll1 and Jagged1). Primary pGCs were transfected with NR5A2 siRNA (siNR5A2), NR2F2 siRNA (siNR2F2), or scramble siRNA (siNC). ( B ) Schematic representation of the Notch/NR5A2/NR2F2 pathway in regulating steroidogenesis pGCs. * means significant difference at p < 0.05.

Article Snippet: The proteins were subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany) and probed with primary antibodies against StAR (DF6192, Affinity Biosciences, Changzhou, China), Cyp11a1 (DF6569, Affinity Biosciences), HSD3B (DF6639, Affinity Biosciences), ERK1/2 (4695T, Cell Signaling Technology, Danvers, MA, USA), pERK1/2 (4370T, Cell Signaling), NR2F2 (NBP1-31980, Novus Biologicals, CO, USA), NR5A2 (NBP2-27196SS, Novus) and β-actin (20536-1-AP, Protein Tech, Wuhan, China).

Techniques: Knockdown, Expressing, Transfection

Journal: Nature communications

Article Title: An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

doi: 10.1038/s41467-023-36967-2

Figure Lengend Snippet: Fig. 2 | NR2F2-Iso2 promotes anchorage-independent growth and melanoma sphere formation in vitro. a qRT-PCR showing relative NR2F2-Iso1 and NR2F2-Iso2 transcript levels normalized to SK-MEL-147, where NR2F2-Iso2 is partly methylated (M) and unmethylated (U). Methylation status was determined with Illumina 450 K arrays and/or methylation specific PCR. b Western blots of melanoma cell lines probed with Isoform-specific NR2F2 antibodies. c Heatmap illustrating CpG- methylation status of NR2F2-Iso2 based on Illumina 450 K array data. d Pie charts showing % CpG methylation of indicated sites at the NR2F2 locus as determined by bisulfite sequencing. Asterisks denote CpGs interrogated in 450 K arrays. e qRT- PCR (left) and western blotting (right) show that treatment of MeWo cells with the demethylating agent 5-aza-2′-deoxycytidine (2.5 μM aza, 72 h) permits NR2F2-Iso2 expression (bars represent SD). One experiment of 2 biological replicates is shown; further validated in other cell lines in Supplementary Fig. 7. f qRT-PCR and western blotting validate isoform-specific NR2F2-Iso2 depletion in NR2F2-Iso2 expressing 4L cells with shNR2F2-Iso2 (shA, shB) compared to shSCR (control) (bars represent SD). Actin served as loading control. Representative result from one of multiple repeats is shown (>3). g Growth rates of shSCR and shNR2F2-Iso2 expressing 4L

Article Snippet: Lentiviral vectors were also used for NR2F2 ectopic expression: NR2F2 Isoform 1 (EX-C0221-Lv205, GeneCopoeia) and NR2F2 Isoform 2 (RC226609L2, Origene). pEZLv205 (EX-NEG-Lv205, GeneCopoeia) or pLenti-C-mGFP (PS100071, Origene) were used as control vectors.

Techniques: In Vitro, Quantitative RT-PCR, Methylation, Western Blot, CpG Methylation Assay, Sequencing, Expressing, Control

Journal: Nature communications

Article Title: An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

doi: 10.1038/s41467-023-36967-2

Figure Lengend Snippet: Fig. 6 | NR2F2-Iso1 reduces colony formation ability and metastatic potential. a qRT-PCRand b western blotting confirms ectopic NR2F2-Iso1 expression in MeWo cells (bars represent SD). c Bar graph showing significantly reduced colony forming ability of MeWo cells that ectopically express NR2F2-Iso1 28 days after seeding (two-tailed unpaired T test, bars represent SD). d qRT-PCR data and e western blotting showing differences in NR2F2-Iso1 expression between shSCR and shNR2F2-Iso1 (shX, shY) MeWo cells that ectopically express GFP or GFP-NR2F2- Iso2 (bars represent SD). f Bar graphs showing relative colony forming efficiencies of these cells 21 days after seeding (two-way ANOVA, bars represent SD) (n = 6 experimental replicates). Experiments in a–f have been done al least twice; c and f three times. (g–i) 4L cells (n = 9 GFP, 10 Iso1 OE NSG mice) labeled with lentiviruses

Article Snippet: Lentiviral vectors were also used for NR2F2 ectopic expression: NR2F2 Isoform 1 (EX-C0221-Lv205, GeneCopoeia) and NR2F2 Isoform 2 (RC226609L2, Origene). pEZLv205 (EX-NEG-Lv205, GeneCopoeia) or pLenti-C-mGFP (PS100071, Origene) were used as control vectors.

Techniques: Western Blot, Expressing, Two Tailed Test, Quantitative RT-PCR, Labeling