Knockdown efficiency in AWV^Δex mice.

<p>(A) eGFP and tdTomato fluorescent signals in mouse organs. Mouse genotypes (No. 1 to 7) are indicated on the right. Mice 5 to 7 are AWV^Δex/eGFP mice, in which the transgene included in each mouse had been derived from a different founder (F0) mouse (lines 1 to 3). An amiRNA expression cassette in AWV^Δex Tg mice is shown (upper right). (B) eGFP fluorescent signals in spleen cells analyzed by FACS. (C) Quantitative real-time PCR of eGFP mRNA expression in mouse liver and kidney tissues. (D) tdTomato fluorescent signals in spleen cells analyzed by FACS. Numbers 1 to 7 in (B–D) correspond to those shown in (A).</p

Removing the tdTomato marker gene from an amiRNA expression cassette enhances knockdown efficiency.

<p>(A) Schematic representation of the experimental strategy. An amiR-eGFP expression vector (pPBKD-G1) that contained a tdTomato marker gene flanked by <i>rox</i> sites was co-transfected into eGFP-expressing ES cells along with a pPBase and a pPBN. Stably transformed clones that exhibited tdTomato fluorescence (+MG clone) were isolated first. Then, these clones were administrated Dre recombinase to remove the tdTomato gene, which resulted in generating a marker-less clone (−MG clone). (B) eGFP fluorescence intensity in ES cells. The experiments were repeated six times and the representative histogram is shown (left). Cell lines 3 (solid line) and 4 (dot line) were used for analysis. Red and blue lines represent eGFP fluorescence in a “+MG clone” and a “−MG clone,” respectively. Green and black lines indicate eGFP fluorescence intensity in eGFP-expressing and wild-type (eGFP-negative) ES cells, respectively. The box plots show the normalized MFI obtained in six experiments (right); *<i>P</i> = 1.8 × 10⁻³, **<i>P</i> = 1.4 × 10⁻⁵ (paired <i>t</i>-test). (C) Quantitative real-time PCR of amiR-eGFP123 (left) and amiR-eGFP419 (right) in cell lines 3 (+MG and −MG) and 4 (+MG and −MG); The experiments were repeated three times; *<i>P</i> = 2.6 × 10⁻³, **<i>P</i> = 6.9 × 10⁻³ (two tailed Student’s <i>t</i>-test). The differences are not significant in amiR-eGFP419 expression (P > 0.05).</p

Comparison of knockdown efficiencies of different amiR-eGFP architectures in eGFP-expressing ES cells.

<p>(A) Structures of amiR-eGFP expression vectors. Two amiRNAs, each of which was designed to target different positions of the eGFP gene (hairpin structures in pink, for eGFP123, and blue, for eGFP419), were used (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135919#pone.0135919.s008" target="_blank">S1 Table</a> for details). (B–D) Fluorescence intensity determined after transfecting eGFP-expressing ES cells with each vector (No. 1 to 6 in A). At 2 days after transfection, the fluorescence intensity of eGFP (B and C) and tdTomato (D) was assessed by FACS. Orange lines (arrowheads in B and C) are eGFP fluorescence intensities of non-transformed eGFP-expressing ES cells (control: C). The mean fluorescence intensities (MFIs) are indicated in the upper right-hand corner of each graph in (B–D). Numbers 1 to 6 in (B–D) correspond to those shown in (A).</p

Generation of marker gene-less (−MG) amiR-eGFP-expressing mice.

<p>(A) Schematic representation of the strategy used to eliminate extra sequences in AWK mice. Extra sequences including vector sequence (shown in moss green line) and an FLPe expression cassette in the ex-allele, were removed by administering FLPe, which resulted in generating an Δex-allele. An FLPe expression cassette in FLPe Tg mice is shown below. Red arrows indicate primer-binding sites used for genotyping analysis. (B) Genotyping results of pups obtained by <i>in vitro</i> fertilization using frozen sperm derived from an AWK^ex/Δex/FLPe mouse (lanes 1 to 20). Lane 21: negative control. PCR conducted using the primer set shown in (A) was used to assess the presence of transgenes. Fifteen individuals (1 to 15) were alive, whereas 5 pups (16 to 20) died just after birth [Note: for the sample No. 20 with primer set (M632/M244), the PCR product was loaded onto a separate lane in the same gel and rearranged in this Figure for clarity]. (C) Body weights of littermates obtained by crossing an AWK^ex mouse with an FLPe Tg mouse. The AWK^ex/Δex/FLPe mouse contained both type of cells (with the AWK^Δex allele or AWK^ex allele) classified as a mosaic mouse. (D) Cataracts developed in the AWK^ex/Δex/FLPe mouse. All mice, similar to those observed in (C), were examined at 66 weeks of age. Cataract phenotypes in additional AWK^ex/Δex/FLPe mice are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135919#pone.0135919.s006" target="_blank">S5 Fig</a>. (E) Scatter plot of quantitative real-time PCR of amiR-eGFP123 expressions (log2 of normalized data) in nine mosaic mice and one AWK^ex mouse including littermates as in (D). Each circle and triangle represents an individual mouse. The values are expressed as mean ± SD represented by the green line for mosaic mice.</p

Michael Addition–Aromatization Reaction of Dienylimines Bearing a Leaving Group and Its Application to the Preparation of Thiol-Selective Labeling Reagents Capable of Forming Strong Carbon–Sulfur Bonds

The reaction of a dienylimine with thiols was found to proceed smoothly to afford the corresponding indolines bearing aromatic carbon–sulfur bonds as a result of a Michael addition–aromatization sequence. Furthermore, this reaction was applied to the development of fluorogenic dienylimines that could be used as thiol-selective fluorescent labeling reagents

Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs

<div><p>The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen <i>(SLA)-1</i>*<i>0401</i> after the stimulation of peripheral blood mononuclear cells (PBMCs) by <i>Staphylococcus aureus</i> superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only <i>SLA-1</i>*<i>0401</i>, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from <i>SLA-1</i>*<i>0401</i> homozygous pigs were stimulated, the <i>SLA-1</i>*<i>0401</i> mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the <i>SLA-1</i>*<i>0401</i> mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.</p></div

Tertiary structure of X2F6 mAb and the predicted antibody epitope.

<p>(A) Amino acid sequences of heavy and light chains of the X2F6 variable region. The database sequence PDB ID 3V7A is shown as the control sequence. (B) The predicted tertiary structure of the X2F6 mAb. (C) The tertiary structures of the YLL set in SLA-1*0501, which reacts with X2F6 with high reactivity (left panel), and the DVF set in SLA-1*1104, which cannot react with X2F6 (right panel), are shown. Pink (hydrophobic) and green (hydrophilic) colors represent the amino acid character. The structure is largely different, and the binding affinity is predicted to be different.</p

Primer sequences.

<p>Primer sequences.</p

Class I SLA-related mRNA expression after TSST-1 stimulation.

<p>The PBMCs of two pigs with the Hp-16.0 haplotype (individuals #965 and #1938) were examined for classical class I SLA (A) and related mRNA (B) expression after stimulation. Closed squares with a solid line show TSST-1-stimulated PBMCs, open squares with a broken line show IFN-γ stimulation, and closed squares with a dotted line show the negative control.</p

Specificity of the X2F6 mAb.

<p>(A) <i>SLA-1</i>*<i>0401</i>, <i>SLA-2</i>*<i>0901</i>, and <i>SLA-3</i>*<i>0602</i>, which are the classical class-I SLA alleles of Haplotype Hp-16.0, and <i>SLA-6</i>*<i>0101</i>, which is a non-classical class-I SLA allele of Hp-16.0, were transfected into HEK293 parent cells, and the reactivity of X2F6 (right panels) was examined by flow cytometry (FCM). Propidium iodide (PI) positive-dead cells were avoided for the gating. PT-85A, the pan-specific MHC class-1 antibody, was used for the positive control (middle panels). (B) The species specificity was examined using swine (Hp-16.0), human, and common marmoset PBMCs. Lymphoid gate was used for the analysis. The percentages shown above the panels are the double-positive cell percentages.</p