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. 2018 Apr 13;9:1466. doi: 10.1038/s41467-018-03731-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Bcan-Ntrk1 gene fusion drives high-grade glioma formation. a Schematic representation of the Bcan and Ntrk1 gene loci and the Bcan-Ntrk1 gene fusion. Indicated are the gRNAs targeting both genes and the primers used for the PCR amplification of the indicated genomic regions. b Top panel: RCAS-gRNA-pair vector expressing the Bcan and Ntrk1 gRNAs. Bottom panels: PCRs were performed with the specified primers on genomic DNA extracted from the p53-null TVA-Cas9 NSCs transduced with the indicated gRNAs. The PCR band for the Bcan-Ntrk1gRNA infected cells was sub-cloned and analyzed by Sanger sequencing. The sequences of four independent clones and a representative chromatogram are shown. c Left panel: Diagram of fluorescence in situ hybridization (FISH) probe design. BAC clone BMQ-437D10 (red) is located within the deleted region and BMQ-386N22 (green) is used as a control of chromosome 3. Mouse BACs are represented as green and red bars. Right panels: Representative FISH results using the two-color probe designed to detect the Ntrk1-Bcan intergenic microdeletion. The control green signal was used to count the number of chromosomes 3. The loss of the red signals indicates the microdeletions. Scale bar: 5 μm. d Top panel: Schematic representation of Bcan-Ntrk1 fusion transcript. Bottom panels: (left) RT-PCRs were performed on the mRNA from the p53-null TVA-Cas9 NSCs transduced with the indicated gRNAs, using the Bcan-Fw and Ntrk1-Rev primers; (middle) the PCR band was sub-cloned and the sequences of two independent clones and a representative chromatogram are shown; (right) qPCR, with the Ntrk1_3′-Fw and Ntrk1_3′-Rev primers, showing the upregulation of the Ntrk1 mRNA in the cells expressing the Bcan-Ntrk1 fusion. Data presented as mean ± SD (n = 3); A.U. arbitrary unit. e H&E and IHCs using the indicated antibodies. White arrows point to mitotic figures. Scale bars: H&E 100 μm; IHCs 50 μm. f Kaplan Meier survival curves of RCAS-Bcan-Ntrk1-gRNA pair-induced gliomas generated in Gtv-a; hGFAP-Cre; LSL-Cas9; p53^lox/lox injected mice. Tva⁻ or Cre⁻ control littermates were used as injection controls. Log-rank P value = 0.0008. g A PCR performed on genomic DNA extracted from Bcan-Ntrk1 tumor tissues (lanes 2–4) confirmed the presence of the Bcan-Ntrk1 gene fusion. Genomic DNA from NSCs was used as a negative control (lane 1)