9 research outputs found

    Gene Transfer and Genome-Wide Insertional Mutagenesis by Retroviral Transduction in Fish Stem Cells

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    <div><p>Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.</p></div

    Genome-wide distribution of retroviral insertions.

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    <p>(A) Cloning and sequencing of insertion events by LM-PCR. MlucI digests of genome DNA was amplified by 5′-biotin-labeled primer for synthesis of ssDNA. The purified ssDNA was ligated with linker and subsequently amplified via two rounds PCR by using primers P1 plus LTR-rev1 and P2 plus LTR-rev2 and cloned for sequencing. (B) Chromosomal distribution of the 350 insertion events. vertical line, chromosome; horizontal bar, site of integration; color; genomic location. (C) Percent genomic locations of 350 insertion events. (D) Junction sequences of representative insertion events.</p

    Retention of ES cell property.

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    <p>Pure populations of GFP-expressing transgenic HX1 cells were obtained by derived rvGTgfp transduction and FACS. Cells in three groups (HX1 control, HX1 transgenics and control + transgenics at a ratio of 1:1) were induced differentiation by EB formation maintained in suspension culture. EBs were directly photographed or stained with hoechst33342, squashed in slide and observed. transgenic HX1 cells(green), parental HX1 (unlabeled). Scale bar, 100 μm (Fig A, B, C), 25 μm (Fig A’, A”, B’, B”, C’, C”). (A, B, C) Three groups of cells were able to form typical EBs. Ebs were photograhped under phase contrast and GFP. (A’, B’, C’) EBs squashes under closer inspection of phase contrast and GFP. (A”, B”, C”) EBs squashes under closer observation of hoechst33342 and GFP. (J) Expression of pluripotency gene and differentiation genes in parental HX1 and transgenic HX1 before and after induced differentiation. Numbers of PCR cycles are indicated to the right. Genes chosen are markers for pluripotency (nanog) and differentiated lineages (nf200, ectoderm; ntl, mesoderm; sox17, endoderm). β-actin served as a loading control.</p

    Cotransduction of SG3 cells.

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    <p>Medaka spermatogonia cells SG3 were infected with rvLcherry alone or plus rvLegfp at MOI of 50 and photographed at 3 dpi. (A and B) SG3 after rvLcherry infection. (C-F) SG3 after infection with rvLcheery and rvLegfp. Scale bars, 100 μm.</p

    Gene trapping in haploid medaka ES cell line HX1.

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    <p>Medaka haploid ES cell line HX1 was infected with rvLcherry and rvGTgfp at MOI = 50 each and observed for gene trapping (green) and random gene insertion (red) at 3 dpi. (A) Consequence of retroviral integration on reporter expression. Any insertions by rvLcherry are predicted to be productive for Cherry expression due to presence of the strong promoter CV. Only a minority of insertions by rvGTgfp is productive for GFP expression due to absence of a promoter. For more details on reporter expression cassettes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127961#pone.0127961.g001" target="_blank">Fig 1A</a>. (B) Phase contrast micrograph, showing cell density and phenotype. (C) Cherry micrograph, showing Cheery expression from random insertions. (D) GFP micrograph, showing GFP expression from gene trapping. (E) Merged fluorescent micrograph. Scale bars, 100 μm.</p

    Retroviral vectors and transduction in mouse and medaka cells.

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    <p>(A) Maps of plasmids pLegfp, pLcherry and pGTgfp for retroviruses rvLegfp, rvLcherry and rvGTgfp. CV, human cytomegalovirus early gene enhancer/promoter; LTR, long terminal repeat; cherry, gene for cherry fluorescent protein; gfp, gene for green fluorescent protein; neo, neomycin phosphotransferase gene; psi, packaging element (ψ); SA, splicing acceptor. (B-F) Micrographs of rvLegfp-infected cells. Cells were infected at MOI = 50 and photographed at 3 dpi by using fluorescent (left panel) and phase-contrast optics (right panel). Average percentage values of GFP-positive cells (green) derived by cell counting in three independent experiments are shown. (B) NIH/3T3 as the positive control. (C) MES1. (D) HX1. (E) SG3. (F) Medaka fibroblasts in primary culture (MF). Scale bars, 100 μm.</p

    Retroviral transduction efficiency in medaka cells.

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    <p>(A) Dose-dependent transduction efficiency of rvLegfp in fish cell lines. Cells were infected with rvLegfp at indicated MOI and the transduction efficiency was determined by GFP expression by counting at least 1000 cells at 3 dpi. (B) Titer of rvLegfp in fish cell lines. Cells were transduced with rvLegfp at MOI of 50 and titer was determined at 3 dpi. Data are means ± SD from three independent experiments. 3T3, mouse fibroblast cell line NIH/3T3 used as the positive control; MES1, medaka diploid ES cell line; HX1, medaka haploid ES cell line; MF, medaka fibroblast cells; SG3, medaka spermatogonial cell line.</p

    Transcription Factor Response Elements on Tip: A Sensitive Approach for Large-Scale Endogenous Transcription Factor Quantitative Identification

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    The ability to map endogenous transcription factors (TFs) DNA binding activity at the proteome scale will greatly enhance our understanding of various biological processes. Here we report a highly sensitive, rapid, and high-throughput approach, transcription factor response elements on tip-mass spectrometry (TOT-MS), that allows for quantitative measurement of endogenous TFs. A total of 150 TFs from 1 μg of nuclear extracts can be quantified with single shot mass spectrometry detection in 1 h of machine time. Up to 755 TFs, which is comparable to the depth of RNA-seq, were identified by TOT coupled with on-tip small size reverse-phase liquid chromatography. We further demonstrated the capability of TOT-MS by interrogating the dynamic change of TFs in the epidermal growth factor (EGF) signaling pathway. This approach should find broad applications in elucidating the TF landscape from limited amounts of biological materials

    A multi-organ nucleus segmentation challenge

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    Generalized nucleus segmentation techniques can contribute greatly to reducing the time to develop and validate visual biomarkers for new digital pathology datasets. We summarize the results of MoNuSeg 2018 Challenge whose objective was to develop generalizable nuclei segmentation techniques in digital pathology. The challenge was an official satellite event of the MICCAI 2018 conference in which 32 teams with more than 80 participants from geographically diverse institutes participated. Contestants were given a training set with 30 images from seven organs with annotations of 21,623 individual nuclei. A test dataset with 14 images taken from seven organs, including two organs that did not appear in the training set was released without annotations. Entries were evaluated based on average aggregated Jaccard index (AJI) on the test set to prioritize accurate instance segmentation as opposed to mere semantic segmentation. More than half the teams that completed the challenge outperformed a previous baseline. Among the trends observed that contributed to increased accuracy were the use of color normalization as well as heavy data augmentation. Additionally, fully convolutional networks inspired by variants of U-Net, FCN, and Mask-RCNN were popularly used, typically based on ResNet or VGG base architectures. Watershed segmentation on predicted semantic segmentation maps was a popular post-processing strategy. Several of the top techniques compared favorably to an individual human annotator and can be used with confidence for nuclear morphometrics
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