Characterization of cANE activity as an early neural plate specific enhancer.

<p>(A-I,A’-I’) Compared expression patterns of <i>cyp26a1</i> (A-I) and <i>egfp</i> driven by cANE (A’-I’) during early embryonic development. (J) Double in situ hybridization showing <i>barhl2</i> expression domain (blue) exactly filling the gap in the <i>cyp26a1</i> expression domain (red). (A,B,C,E,G,A’,B’,C’,E’,G’) are lateral views. (d,f,h,I,D’,f’,h’,I’) are animal pole views. White arrowheads: anterior neural plate. Black arrowheads: blastoderm marginal zone. Arrows in (H-J): gap in the anterior neural plate domain of <i>cyp26a1</i> expression. All stages are indicated in the pictures. (k,k’,l,l’) The effect of 100 nM retinoic acid (RA) treatment between 2,5 hpf and 8,5 hpf on on stable transgenic cANE_endo:::<i>egfp</i> (K-K’) and cANE::<i>egfp</i> (L-L’) expression. (M) EGFP fluorescence in a 12 hpf stable transgenic cANE:::<i>egfp</i> embryo. Lateral view with dorsal to the left. (N) Schematic representation of cANE and all three reported retinoic acid responsive elements (R1, R2, R3) identified previously. cANE is located from -504 bp to -195 bp relative to <i>cyp26a1</i> ATG codon. An: animal, Vg: vegetal; V: ventral; D: dorsal, A: anterior, P: posterior, L: left, R: right, CTRL: control embryo, RA: retinoic-acid treated embryo.</p

Detailed dissection of cANE by transient expression of deletion constructs in zebrafish embryos.

<p>(A) Schematic view of the constructs used for dissection of the cANE module. All constructs contain one single fragment (black) from the cANE module, placed immediately upstream of the <i>gata2</i> minimal promoter, except for construct cANE_endo, where the <i>gata2</i> promoter is replaced by the <i>cyp26a1</i> endogenous promoter. Numbers correspond to nucleotide coordinates within the 310 nt zebrafish cANE. The hatched block (Motif1) represents the 12-bp difference between constructs 39–310 and 50–310; the highly conserved blocks are indicated by checkered patterns. (B-K) Enhancer activity of the cANE deletions shown in (A), assayed by <i>egfp</i> in situ hybridization in transient or stable (labelled Tg()) transgenic embryos. All embryos are between stages 10.5 and 11 hpf, viewed from the animal pole, with anterior on the left.</p

Conservation of vertebrate <i>cyp26a1</i> promoter region with zebrafish cANE.

<p>Alignment of 4 teleostean (tetraodon, fugu, stickleback and medaka) and mouse and human <i>cyp26a1</i> promoter regions orthologous to zebrafish cANE. The most conserved regions are outlined with red (predicted SoxB binding sites 2 and 3) or purple (conserved block1,2,3) boxes. The green (Motif1) and blue (predicted SoxB binding site 1) boxed sequences lie in the non-evolutionarily conserved region of zebrafish cANE. Numbers correspond to nucleotide coordinates within the 310 nt zebrafish cANE.</p

Role of SoxB binding sites in the regulation of <i>cyp26a1</i> expression in the anterior neural plate (ANP).

<p>(A) Representation of the cANE region in the zebrafish genome; 3 predicted SoxB binding sites are indicated; the green channel represents the intensity of the anti-Sox2 ChipSeq signal in this region according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150639#pone.0150639.ref034" target="_blank">34</a>]. (B) A logo representing the composite consensus binding site for SoxB/Oct factors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150639#pone.0150639.ref033" target="_blank">33</a>] is aligned with the predicted Sox binding site (Sox_BS1) overlapping Motif1 (cANE 45–59). The mutSox TT->CC mutation destroying the Sox-binding half-site is indicated. (C-F) egfp in situ hybridization representing enhancer activity of cANE ΔSoxBS2-3, where both Sox_BS2 and Sox_BS3 have been deleted (C), compared with intact cANE (D), at the 90% epiboly stage, and enhancer activity of Motif1 mutation mutSox (F) in 1–222 context compared with wild type 1–222 (E), at the 75% epiboly stage. Dorsal views; anterior is to the left. (G) RT-PCR relative quantification of total <i>egfp</i> expression stable transgenic embryos for constructs cANE (1–310), 81–310, 1–222 and 1–222 mutSox, as well as non transgenic embryos (Ctl); error bars represent SEM; units are arbitrary.</p

Additional file 3: Figure S1. of Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

Overlap of off-target detection for the EMX1 and VEGFA guides tested by different assays. Off-targets are only shown if they were detected by at least a single study and with a frequency of 0.1 %. See Additional file 1: Tables S1 and Additional file 4: Table S2 for the modification frequencies and additional details on the off-targets for the guides EMX1 and VEGFA, respectively. Additional file 4: Table S2 also includes the data by Hsu et al. [7], who quantified cleavage at putative off-target loci predicted by the CRISPR Design website ( http://crispr.mit.edu/ ) with targeted deep sequencing, Tsai et al. [3], who isolated double-strand breaks with modified oligonucleotides followed by sequencing, Frock et al. [28], who detected translocations, and Kim et al. [33] and Kim et al. [27], who performed whole-genome sequencing to find CRISPR-induced modifications. For details on the different studies, see Additional file 1: Table S1. (PDF 17 kb

Ilex serrata Thunb.

原著和名: ウメモドキ科名: モチノキ科 = Aquifoliaceae採集地: 愛知県 豊橋市 岩崎町 (三河 豊橋市 岩崎)採集日: 1968/10/20採集者: 萩庭丈壽整理番号: JH042317国立科学博物館整理番号: TNS-VS-99231

Mentha viridis L.

原著和名: ミドリハクカ科名: シソ科 = Labiatae採集地: 千葉県 千葉市 千葉大学 (下総 千葉市 千葉大学)採集日: 1967/8/13採集者: 萩庭丈壽整理番号: JH042898国立科学博物館整理番号: TNS-VS-99289

Acanthopanax trichodon Franch. et Savat.

原著和名: ミヤマウコギ科名: ウコギ科 = Araliaceae採集地: 千葉県 清澄山 (上総〜安房 清澄山)採集日: 1961/6/18採集者: 萩庭丈壽整理番号: JH042319国立科学博物館整理番号: TNS-VS-99231