Nuclear localization of CT311 in <i>C.</i><i>trachomatis</i>-infected cells.

<p>(A) HeLa cells infected with <i>C. trachomatis</i> organisms were processed at different time points after infection (as indicated on top of the image) for immunofluorescence labeling of chlamydial organisms (green), CT311 (panels a to e) or CPAF (panels f to j, both red) or DNA (blue). Red arrowheads indicate CT311 localized in the host cell nuclei in samples harvested at 36h (d) and 44h (e) post infection. The images were acquired using a conventional fluorescence microscope. (B) The 36h sample was further observed under a confocal microscope. Red arrowhead indicates nuclear localization of CT311.</p

Basic amino acids required for targeting GFP into nucleus.

<p>(A) The amino acid sequence covering residue 21 to 63 defined as CT311 fragment 4 (F4) or nuclear localization signal sequence (NLS) was shown with two clusters of basic residues highlighted in red and marked as Clusters 1 & 2 (C1 & C2) respectively. Cluster 2 consists of 2 separate basic residue sub-clusters, designated as C2a & C2b respectively. The sequences of constructs with C2b deletion (C2b del-GFP) and C1 or C2a substitution mutations (C1 sub-GFP or C2a sub-GFP) were also listed. (B) HeLa cells transfected with pLEGFP plasmid alone or expressing the various constructs listed in (A) were processed and observed under a fluorescence microscope as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064529#pone-0064529-g004" target="_blank">Fig. 4B</a> legend. Deletion or substitution of C2b, C1 or C2a effectively blocked the nuclear targeting of GFP by the CT311 NLS. (C) The GFP signal in the nuclei and entire cells was semi-quantitatively measured and the % of nuclear GFP signal from each sample was compared between cell samples as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064529#pone-0064529-g004" target="_blank">Fig4</a>. ** indicates statistically significant differences (P<0.01) and “ns” stands for no significant difference. The data were from 3 independent experiments.</p

Localization of CT311 expressed via a transgene in mammalian cell nuclei.

<p>(A) HeLa cells transfected with pLenti6.3/V5-CT311 (panels a–c) or pLenti6.3/V5-CPAF (panels d to f) were processed 24h after transfection for immunofluorescence labeling of CT311 or CPAF using an anti-V5 tag antibody (red) and DNA using Hoechst dye (blue). CT311 but not CPAF localized in host cell nuclei. (B) The nuclear localization of CT311 was confirmed by co-labeling CT311 (green) and Nup153 (red), a nuclear protein.</p

Summary of the identification of the yellow catfish carrying disrupted <i>mstn</i>.

<p>Summary of the identification of the yellow catfish carrying disrupted <i>mstn</i>.</p

Zebrafish embryos can be used as an <i>in vivo</i> system to examine ZFN activity of editing yellow catfish <i>mstn</i> gene.

<p>(A) Schematic diagram shows ZFN1 binding to the yellow catfish <i>mstn</i> gene. Yellow catfish <i>mstn</i> exons are shown as boxes and its introns are shown as solid lines. Start codon (ATG) and stop codon (TGA) are marked in exon 1 and exon 3, respectively. Number above the exon box denotes the position of nucleotides in the gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028897#pone.0028897-Pan1" target="_blank">[4]</a>. The ZFN1 binding site is in exon 1. The triplets of nucleotides recognized by ZFN1 fingers are marked in different colors. (B) Zebrafish embryos were used as an <i>in vivo</i> system to examine ZFN activity of editing yellow catfish <i>mstn</i> gene. The plasmid containing exon 1 of yellow catfish <i>mstn</i> gene (pGEM-ycMSTN) was co-microinjected with ZFN1 mRNA into zebrafish embryos at 1–2-cell stage. The <i>mstn</i> molecules were amplified from the zebrafish embryos at 24 hpf and then subcloned for sequencing. Analyses on sequences of the molecules revealed that the molecules of disrupted <i>mstn</i> were categorized into three groups including deletions, insertions and complex. (C) ZFN1 cut <i>mstn</i> in yellow catfish genome. ZFN1 mRNA was microinjected into yellow catfish embryos at 1–2-cell stage. The <i>mstn</i> molecules were amplified from the yellow catfish embryos at 72 hpf and then subcloned for sequencing. Analyses on sequences of the molecules revealed that the molecules of disrupted <i>mstn</i> in yellow catfish genome were categorized into three groups including deletions, insertion and complex. WT: partial sequence of wild type <i>mstn</i> containing ZFN1 targeting site (B, C). Number in the leftmost of the panels (B, C) shows the number of nucleotides was deleted (−) or inserted (+) in the mutated <i>mstn</i> gene. Number in the bracket shows the frequency of the mutated molecules (B, C). Inserted nucleotides are bolded (B, C).</p

Additional file 1: of An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease

Supplemental Table and Figures. Supplemental Table S1 and Figures S1–S7. (PDF 742 kb