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