Turn-on Fluorescent Probe for Exogenous and Endogenous Imaging of Hypochlorous Acid in Living Cells and Quantitative Application in Flow Cytometry

Hypochlorous acid (HClO) acts as a dominant microbicidal mediator in the natural immune system, and the excess production of hypochlorites is related to a series of diseases. Thus, it is vitally important and necessary to develop a highly sensitive and selective method for HClO detection in living systems, and most of fluorescent probes are mainly focused on cells imaging. Besides, accurate HClO quantitative information about individual cells in a large cell population is extremely important for understanding inflammation and cellular apoptosis as well. In our work, a turn-on fluorescent probe has been synthesized, which can selectively and sensitively detect HClO with fast response time. The probe is almost nonfluorescent possibly due to both the spirolactam form of fluorescein and unbridged CN bonds which can undergo a nonradiative decay process in the excited state. Upon the addition of ClO^–, the probe was oxidized to ring-opened fluorescent form and the fluorescence intensity was greatly enhanced. In live cell experiments, the probe was successfully applied to image exogenous ClO^– in HeLa cells and endogenous HClO in RAW 264.7 macrophage cells. In particular, the quantitative information on exogenous and endogenous HClO can also be acquired in flow cytometry. Therefore, the probe not only can image exogenous and endogenous HClO but also provides a new and promising platform to quantitatively detect HClO in flow cytometry

Cell-penetrating peptide-driven Cre recombination in porcine primary cells and generation of marker-free pigs

<div><p>Cell-penetrating peptides (CPPs) have been increasingly used to deliver various molecules, both <i>in vitro</i> and <i>in vivo</i>. However, there are no reports of CPPs being used in porcine fetal fibroblasts (PFFs). The increased use of transgenic pigs for basic research and biomedical applications depends on the availability of technologies for efficient genetic-modification of PFFs. Here, we report that three CPPs (CPP5, TAT, and R9) can efficiently deliver active Cre recombinase protein into PFFs via an energy-dependent endocytosis pathway. The three CPP<b>–</b>Cre proteins can enter PFFs and subsequently perform recombination with different efficiencies. The recombination efficacy of CPP5<b>–</b>Cre was found to be nearly 90%. The rate-limiting step for CPP<b>–</b>Cre-mediated recombination was the step of endosome escape. HA2 and chloroquine were found to improve the recombination efficiency of TAT<b>–</b>Cre. Furthermore, we successfully obtained marker-free transgenic pigs using TAT<b>–</b>Cre and CPP5<b>–</b>Cre. We provide a framework for the development of CPP-based farm animal transgenic technologies that would be beneficial to agriculture and biomedicine.</p></div

Design of expression cassettes, purification of the three CPP–Cre proteins, and assessment of their activities in an <i>in vitro</i> assay.

<p>(A) Schematic description of the three CPPs<b>–</b>Cre expression constructs. All the constructs encode Cre recombinase with a His-tag (represented by blue and green boxes, respectively). Red boxes represent CPPs (RQRRKKRG): R9 (RRRRRRRRR), TAT (YGRKKRRQRRR), and CPP5 (KLVPM). (B-D) SDS-PAGE analysis of the purification of CPP<b>–</b>Cre proteins. M, marker; SF, supernatant fraction; PR, precipitation; Ni, Nickel column; G25, G25 column. (E) Schematic of recombination <i>in vitro</i>. The assay substrate, pDFR, was linearized by digestion with <i>Bsa</i>I and was used to assess the recombinase activity of the purified Cre proteins. (F-H) Activity of CPP<b>–</b>Cre proteins <i>in vitro</i>. Reactions were carried out in a 50-μL volume with 300 ng of pDFR and New England Biolabs Cre buffer. Different amounts of purified Cre were added, and the mixtures were incubated at 37°C for 30 min. The reactions were then split in half and resolved on a 1% agarose gel. Lane 1 kb ladder; lane 2, NEB Cre; lane 3, 50 ng Cre; lane 4, 100 ng Cre; lane 5, 150 ng Cre. Note the appearance of four bands including the recombined circular plasmid (migrating at the 2.6 kb pC), the nicked recombined circular plasmid pC-N, the rejoined 5.7 kb “stuffer fragment” pL, and the 8.3kb pDFR-L.</p

Effects of Lysosomotropic agents on TAT–Cre recombination efficiency and toxicity.

<p>Effects of different concentrations of HA2 (A) and chloroquine (B) on TAT<b>–</b>Cre-mediated recombination of a loxP-<i>Neo</i>-loxP target, using the standard curve method (39). Percentage of living cells (as an indication of potential toxicity) in porcine fetal fibroblasts treated with different concentrations of HA2 (C) and chloroquine (D). Error bars, s.d. (n = 3).</p

Overexpression of Histone Deacetylase 6 Enhances Resistance to Porcine Reproductive and Respiratory Syndrome Virus in Pigs

<div><p>Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically relevant viral pathogens in pigs and causes substantial losses in the pig industry worldwide each year. At present, PRRSV vaccines do not effectively prevent and control this disease. Consequently, it is necessary to develop new antiviral strategies to compensate for the inefficacy of the available vaccines. Histone deacetylase 6 (HDAC6) is an important member of the histone deacetylase family that is responsible for regulating many important biological processes. Studies have shown that HDAC6 has anti-viral activities during the viral life cycle. However, whether <i>HDAC6</i> overexpression enhances resistance to PRRSV in pigs remains unknown. In this study, we used a somatic cell cloning method to produce transgenic (TG) pigs that constitutively overexpress porcine <i>HDAC6</i>. These TG pigs showed germ line transmission with continued overexpression of HDAC6. <i>In vitro</i>, virus-challenged porcine alveolar macrophages (PAMs) overexpressed <i>HDAC6</i>, which suppressed viral gene expression and PRRSV production. <i>In vivo</i>, resistance to PRRSV in TG pigs was evaluated by direct or cohabitation mediated infection with a highly pathogenic PRRSV (HP-PRRSV) strain. Compared with non-TG (NTG) siblings, TG pigs showed a significantly lower viral load in the lungs and an extended survival time after infection with HP-PRRSV via intramuscular injection. In the cohabitation study, NTG pigs housed with challenged NTG pigs exhibited significantly worse clinical symptoms than the other three in-contact groups. These results collectively suggest that HDAC6 overexpression enhances resistance to PRRSV infection both <i>in vitro</i> and <i>in vivo</i>. Our findings suggest the potential involvement of HDAC6 in the response to PRRSV, which will facilitate the development of novel therapies for PRRSV.</p></div

Generation of marker-free Monoallelic <i>MSTN</i> gene knockout pigs using TAT–Cre.

<p>(A) PCR-based genotyping scheme to differentiate the three <i>MSTN</i> alleles: WT, <i>MSTN</i>^+/−with the <i>Neo</i>, and marker-free <i>MSTN</i>^+/−. P3 and P4 primers designed to amplify 788-bp and 370-bp fragments from marker-free pigs. The 788-bp fragment could only be amplified from WT and <i>MSTN</i>^+/- pigs with a 30-s extension time. (B) Identification of marker-free <i>MSTN</i>^+/−piglets using genomic PCR method. WT, wild-type; H₂O, negative control; M^+/-, <i>MSTN</i>^+/−piglets containing <i>Neo</i>; 1<b>–</b>12 are marker-free <i>MSTN</i>^+/−piglets. (C) PCR sequencing analysis of the marker free <i>MSTN</i>^+/−piglets. The 370-bp product is a specific fragment for the marker-free pig genome including one loxP site between P3 and P4. (D) A live marker-free <i>MSTN</i>^+/−pig.</p

CPP–Cre mediated recombination efficiencies in porcine fetal fibroblasts (PFFs).

<p>Structure of the recombination substrate in loxP-Neo-loxP porcine fetal fibroblasts. The loxP-Neo-loxP PFFs, which were generated from the heterozygous <i>MSTN</i>^<i>+/−</i>knockout pig generated previously in our laboratory, contains a single copy of Neo flanked by loxP sites, such that Cre-mediated recombination would remove Neo and the 600 bp fragment cannot be amplified using primers P1 and P2.(B-D) A standard curve for estimating the CPP–Cre recombination frequencies (39).Genomic DNA isolated from cells treated with CPP-Cre were serially diluted in a buffer and subjected to PCR analysis in a reaction volume of 25 μL. A standard curve for the estimation of deletion frequencies was generated. A plasmid containing the sequence of the PCR product corresponding to the genomic recombination induced by Cre was serially diluted in a solution containing genomic DNA isolated from pig fibroblast cells and the diluted samples were subjected to PCR analysis. The intensities of DNA bands corresponding to the recombination event were measured and plotted against the dilution factors. At high values, the band intensities reached a plateau and thus were excluded when the standard curve was plotted. Error bars, s.d. (n = 3).</p

CPP-mediated Cre recombinase transduction efficiency at 4°C and 37°C, and localization of the CPP–Cre proteins.

<p>(A-C) Transduction efficiencies of CPP<b>–</b>Cre proteins at 4°C and 37°C. Flow cytometry analysis of cells treated for 3 h at 37°C and 4°C at the indicated concentrations of CPP<b>–</b>Cre labeled with Alexa Fluor 488. The cells were washed thoroughly with PBS and then trypsinized to remove the extracellular CPP fusion proteins. (D) Confocal images showing endosomal colocalization of CPP<b>–</b>Cre-488 proteins and FM4-64 (fluorescent, general endosomal marker) in live cells. Scale bar, 10 μm.</p

HDAC6 Overexpression enhanced resistance to infection during cohabitation with PRRSV-infected pigs.

<p>(A) The survival curves of the pigs housed with challenged pigs (log rank test; P = 0.153). n = 5 for each group. (B) Rectal temperature curves for the 4 groups housed with challenged pigs. (C) Body weight curves for the 4 groups housed with challenged pigs. The data were analyzed using ANOVA, which revealed that the body weights of the pigs in the NTG/NTG group were significantly lower than the body weights of the other groups after 5 dpi (P<0.05). (D) Analysis of the viral load in the serum samples obtained from pigs in the 4 housed groups at 4 dpi. (E) Antibody positive tests (S/P>0.4) indicating the level of the immune response <i>in vivo</i>. (F) Survival curves for the two groups of pigs that were housed in two separate rooms (log rank test; P = 0.712). (G) Body weight curves for pigs in the two groups that were housed in two separate rooms. The body weights of the pigs housed with NTG pigs were significantly higher than those of pigs housed with TG pigs at 6, 9 and 10 dpi. (H) Rectal temperature curves for the pigs from the two groups that were housed in two separate rooms. The rectal temperatures of the pigs housed with NTG pigs were significantly higher than were those of pigs housed with TG pigs at 2 and 3 dpi. (I) Survival curves for the housed pigs with different genotypes (log rank test; P = 0.046). (J) Rectal temperature curves for the housed pigs with different genotypes. (K) Body weight curves for the housed pigs with different genotypes. The data in panels B, C and D are presented as the mean±SD. The data in panels B, C and D were analyzed using ANOVA. The data in panels G, H, J, and K are presented as the mean±SE. The statistical significance of these data was analyzed using a t-test. *, P<0.05; **, P<0.01.</p

<i>HDAC6</i> Overexpression inhibits viral gene expression and PRRSV production in PAMs.

<p>(A) qRT-PCR analysis of <i>HDAC6</i> expression in PAMs isolated from pigs using the Q-HDAC6-F/R primer pair. <i>HDAC6</i> expression is presented as a ratio relative to the level of <i>GAPDH</i>. The data are presented as the mean±SD from three independent experiments. (B) Western blot analysis of PAMs isolated from F1 pigs. The protein samples were probed with anti-Actub and GAPDH antibodies. (C) qRT-PCR analysis of viral <i>ORF7</i> RNA levels in TG and NTG PAMs that were inoculated with the PRRSV strain CH-1a (MOI = 0.5) for 24, 48 and 72 h. The data represent the results of three independent experiments (mean±SD). (D) qRT-PCR analysis of viral <i>ORF7</i> RNA levels in TG and NTG PAMs that were inoculated with the PRRSV strain JXA1 (MOI = 0.25) for 24 and 48 h. The data are presented relative to the expression of <i>GAPDH</i> mRNA and represent the results of three independent experiments (mean±SD). RNA from NTG PAMs at 24 hpi was used as the reference sample. Statistical significance was analyzed using a t-test. *, P<0.05; **, P<0.01; ***; P<0.001. (E) The supernatant containing PRRSV RNA was analyzed based on absolute quantitative RT-PCR values at the indicated time points. PAMs were infected with PRRSV CH-1a (MOI = 0.05), and the supernatant was collected and used for RNA extraction and absolute qPCR analysis of the virions at 24, 48 and 72 hpi. The data are representative of the results of three independent experiments (mean±SD). Statistical significance was analyzed using Student’s t-test. **, P<0.01; ***, P<0.001.</p