Cre Protein Research Articles

Abstract LB-135: New cancer prevention model using imaging for early detection of mesothelin-expressing cancers

Abstract A major challenge to the discovery and development of novel cancer prevention strategies is the availability of reliable animal models. Key elements for chemoprevention testing in animal include relevance to human cancers, with tumors of similar pathology and genetic abnormalities, the presence of intermediate lesions that resemble human cancer development both histologically and molecularly; and consistent tumor incidence of >60% within less than 6 months. To our knowledge no established animal model responds to these requirements for ovarian cancer prevention. We sought to adapt the Dicerflox/flox-Ptenflox/flox double knockout (DKO) ovarian cancer mouse model (Kim, Matzuk PNAS 2012) for cancer prevention studies, by synchronized induction of the disease with intra-ovary injection of an adenovirus encoding CRE protein (Ad-mCherry-Cre). Premalignant lesions in DKO AdCRE mice were visible by H&E staining after 12 weeks, and evolved into typical serous ovarian cancer by 24 weeks. Histology demonstrated that tumors overexpressed cytokeratin 8, cyclin D, CA125, and mesothelin. In addition, CA125, the main biomarker for ovarian cancer in woman, was consistently elevated in the serum of DKO AdCRE mice 15 weeks after CRE induction, but not in age-matched DKO mice. To assess whether mesothelin-targeted imaging could help monitor cancer development in DKO AdCRE mice, we used an anti-mesothelin recombinant antibody derived from llama (G3a nanobody) that we previously identified and in vitro characterized (Prantner, Scholler, JBN 2015). G3a nanobody is conveniently secreted by yeast with an N-terminal biotinylation (Scholler, JIM 2006) for streptavidin coupling. We addressed here whether G3a nanobody could detect mesothelin-expressing tumors using in vivo imaging (IVIS). Ni-purified G3a nanobody was incubated with fluorescently-labelled streptavidin (IRB680W) for 30 min and injected retro orbitally just before imaging. We found that G3a nanobody generated a robust and mesothelin-specific signal that peeked 4 hours after injection, and detected DKO AdCRE tumors as early as 8 weeks after CRE induction. G3a nanobody specific detection of mesothelin was confirmed in other mouse models of cancer, including in syngeneic models of ovarian cancer induced by orthotopic or intraperitoneal injection of mesothelin+ Luc-ID8 cells, and in xenograft models of human lung cancer obtained by IV injection of mesothelin high EKVX, mesothelin int H460, and mesothelin lo A549 cells. In summary, our results indicate that G3a nanobody is a valuable reagent for early detection of mesothelin-expressing cancer, and that DKO mice induced for Pten and Dicer double knockout by intra ovarian injection of CRE adenovirus develop serous ovarian cancer that express CA125 and mesothelin. We propose this model for in vivo monitoring of cancer development, as a novel system model for ovarian cancer prevention studies. Citation Format: Khushboo Sharma, Catherine Yin, Kalika Kamat, Claire Repellin, Lidia C. Sambucetti, Nathalie Scholler. New cancer prevention model using imaging for early detection of mesothelin-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-135. doi:10.1158/1538-7445.AM2017-LB-135

Epigenetic upregulation of CXCL12 expression mediates antitubulin chemotherapeutics-induced neuropathic pain.

Clinically, Microtubule-targeted agents-induced neuropathic pain hampers chemotherapeutics for patients with cancer. Here, we found that application of paclitaxel or vincristine increased the protein and mRNA expression of CXCL12 and frequency and amplitude of miniature excitatory post synaptic currents (mEPSCs) in spinal dorsal horn neurons. Spinal local application of CXCL12 induced the long-term potentiation of nociceptive synaptic transmission and increased the amplitude of mEPSCs. Inhibition of CXCL12 using the transgenic mice (CXCL12) or neutralizing antibody or siRNA ameliorated the mEPSC's enhancement and mechanical allodynia. In addition, paclitaxel and vincristine both could increase the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the acetylation of histone H4 in the CXCL12-expressing neurons. Immunoprecipitation and chromatin immunoprecipitation assays demonstrated that antitubulin chemotherapeutics increased the binding of STAT3 to the CXCL12 gene promoter and the interaction between STAT3 and p300, and contributed to the enhanced transcription of CXCL12 by increasing the acetylation of histone H4 in CXCL12 gene promoter. Inhibition of STAT3 by intrathecal injection of adeno-associated virus encoding Cre and green fluorescent protein into STAT3 mice or inhibitor S3I-201 into rats suppressed the CXCL12 upsurge by decreasing the acetylation of histone H4. Finally, blockade of CXCR4 but not CXCR7 ameliorated the paclitaxel- or vincristine-induced mechanical allodynia. Together, these results suggested that enhanced interaction between STAT3 and p300 mediated the epigenetic upregulation of CXCL12 in dorsal horn neurons, which contributed to the antitubulin chemotherapeutics-induced persistent pain.

Nanoparticle-Mediated Recombinase Delivery into Maize.

We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants. Using the experimental procedures and conditions described here, as high as 20% of bombarded embryos can produce regenerable recombinant callus events. This nanomaterial-mediated, DNA-free methodology has potential to become an effective tool for plant genome editing.

Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis.

An approach for restoring recombination activity of complementation split-Cre was developed to excise the transgene in hybrid progeny of GM crops. Growing concerns about the biosafety of genetically modified (GM) crops has currently become a limited factor affecting the public acceptance. Several approaches have been developed to generate selectable-marker-gene-free GM crops. However, no strategy was reported to be broadly applicable to hybrid crops. Previous studies have demonstrated that complementation split-Cre recombinase restored recombination activity in transgenic plants. In this study, we found that split-Cre mediated by split-intein Synechocystis sp. DnaE had high recombination efficiency when Cre recombinase was split at Asp232/Asp233 (866bp). Furthermore, we constructed two plant expression vectors, pCA-NCre-In and pCA-Ic-CCre, containing NCre866-In and Ic-CCre866 fragments, respectively. After transformation, parent lines of transgenic Arabidopsis with one single copy were generated and used for hybridization. The results of GUS staining demonstrated that the recombination activity of split-Cre could be reassembled in these hybrid progeny of transgenic plants through hybridization and the foreign genes flanked by two loxP sites were efficiently excised. Our strategy may provide an effective approach for generating the next generation of GM hybrid crops without biosafety concerns.

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc.

Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels.

The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGATflox/flox mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT−/− mice were the same as Cx57-VGAT+/+ controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT−/− mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT−/− mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors.

Ins1(Cre) knock-in mice for beta cell-specific gene recombination.

Aims/hypothesisPancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cells and their impact on whole body glucose homeostasis. Several Cre recombinase (Cre) deleter mice have been established to allow inactivation of genes in beta cells, but many show non-specific recombination in other cell types, often in the brain.MethodsWe describe the generation of Ins1 Cre and Ins1 CreERT2 mice in which the Cre or Cre-oestrogen receptor fusion protein (CreERT2) recombinases have been introduced at the initiation codon of the Ins1 gene.ResultsWe show that Ins1 Cre mice induce efficient and selective recombination of floxed genes in beta cells from the time of birth, with no recombination in the central nervous system. These mice have normal body weight and glucose homeostasis. Furthermore, we show that tamoxifen treatment of adult Ins1 CreERT2 mice crossed with Rosa26-tdTomato mice induces efficient recombination in beta cells.Conclusions/interpretationThese two strains of deleter mice are useful new resources to investigate the molecular physiology of pancreatic beta cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3468-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Genomic DNA recombination with cell‐penetrating peptide‐tagged cre protein in mouse skeletal and cardiac muscle

The Cre-loxP recombination system has been used to promote DNA recombination both in vitro and in vivo. For in vivo delivery, Cre expression is commonly achieved through the use of tissue/cell type-specific promoters, viral infection, or drug inducible transcription and protein translocation to promote targeted DNA excision. The development of cell permeable (or penetrating) peptide tagged proteins has facilitated the delivery of Cre recombinase protein into cells in culture, organotypic slide culture, or in living animals. In this report, we generated bacterially expressed, his-tagged Cre protein with either a cardiac targeting peptide or an antennapedia peptide at the C-terminus and demonstrated efficient uptake and recombination in both cell culture and mice. To facilitate delivery to cardiac and skeletal muscle, we mixed proteins with pluronic F-127 hydrogel and delivered Cre protein into reporter Rosa26mTmG mouse skeletal muscle or Rosa26LacZ cardiac muscle via ultrasound guided injection. Activation of reporter gene expression indicated that these Cre proteins were enzymatically active. Recombination events were detected only in the vicinity of injection areas. In conclusion, we have developed a method to deliver enzymatically active Cre protein locally to skeletal muscle and cardiac muscle that may be adapted for use with other proteins.

Tissue-specific tolerance of endogenous self antigen-specific CD4+ T cells (IRC4P.484)

Abstract CD4+ T cells specific for self antigens are involved in autoimmunity, but little is known about the extent to which these potentially harmful T cells develop within a normal T cell repertoire or how they are maintained in a tolerant state. We modeled Cre recombinase as a neo-self antigen that is expressed by various different tissue-specific promoters in several strains of Cre transgenic mice. We mapped a CD4+ T cell epitope within the Cre protein and generated a corresponding peptide:MHCII tetramer, which we used in a magnetic enrichment strategy to directly detect rare populations of endogenous Cre-specific CD4+ T cells that naturally develop in the unmanipulated immune systems of these mice. We found that deletion of Cre-specific T cells was efficient in mice ubiquitously expressing Cre, but almost nonexistent in mice expressing Cre in tissue-restricted patterns. In mice expressing Cre exclusively in the pancreas, immunization with Cre antigen induced strong Cre-specific T cell expansion and cytokine production, suggesting immune ignorance to the pancreatic self antigen. In contrast, tolerance to Cre self antigen expressed in mucosal tissues such as the gut and lung relied on elevated steady state frequencies of Cre-specific regulatory T cells. While these Tregs efficiently controlled primary autoimmune responses, their ability to maintain tolerance was lost over successive antigenic challenges, indicating limits of peripheral tolerance to tissue-restricted self antigens.

Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells

Integrating viruses represent robust tools for cellular reprogramming; however, the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here, we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies.

AKI after Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overexpression of stanniocalcin-1 in mice protects against ischemia-reperfusion kidney injury. We sought to determine the kidney phenotype after kidney endothelium-specific expression of stanniocalcin-1 small hairpin RNA (shRNA). We generated transgenic mice that express stanniocalcin-1 shRNA or scrambled shRNA upon removal of a floxed reporter (phosphoglycerate kinase-driven enhanced green fluorescent protein) and used ultrasound microbubbles to deliver tyrosine kinase receptor-2 promoter-driven Cre to the kidney to permit kidney endothelium-specific shRNA expression. Stanniocalcin-1 mRNA and protein were expressed throughout the kidney in wild-type mice. Delivery of tyrosine kinase receptor-2 promoter-driven Cre to stanniocalcin-1 shRNA transgenic kidneys diminished the expression of stanniocalcin-1 mRNA and protein throughout the kidneys. Stanniocalcin-1 mRNA and protein expression did not change in similarly treated scrambled shRNA transgenic kidneys, and we observed no Cre protein expression in cultured and tyrosine kinase receptor-2 promoter-driven Cre-transfected proximal tubule cells, suggesting that knockdown of stanniocalcin-1 in epithelial cells in vivo may result from stanniocalcin-1 shRNA transfer from endothelial cells to epithelial cells. Kidney-specific knockdown of stanniocalcin-1 led to severe proximal tubule injury characterized by vacuolization, decreased uncoupling of protein-2 expression, greater generation of superoxide, activation of the unfolded protein response, initiation of autophagy, cell apoptosis, and kidney failure. Our observations suggest that stanniocalcin-1 is critical for tubular epithelial survival under physiologic conditions.

Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles.

Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision,

The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified "nontransgenic" plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method.

Sox9 Induction, Ectopic Paneth Cells, and Mitotic Spindle Axis Defects in Mouse Colon Adenomatous Epithelium Arising From Conditional Biallelic Apc Inactivation

We generated transgenic mice in which human CDX2 gene elements control expression of a tamoxifen-regulated Cre protein (CDX2P-CreER(T2)) to allow for inducible gene targeting in intestinal epithelium. After tamoxifen dosing of CDX2P-CreER(T2) mice, Cre activity was detected in the distal ileal, cecal, colonic, and rectal epithelium, with selected crypt base, transit amplifying, and surface cells all capable of activating Cre function. Four weeks after tamoxifen dosing of CDX2P-CreER(T2) mice carrying a Cre-activated fluorescent reporter, single crypts were uniformly fluorescence positive or negative, reflecting Cre activation in crypt stem cells. Biallelic inactivation of the Apc tumor suppressor gene via the CDX2P-CreER(T2) transgene in colon epithelium led to acute alterations in cell proliferation, apoptosis, and morphology, along with mitotic spindle misorientation, β-catenin nuclear localization, and induction of the intestinal stem cell markers Lgr5 and Musashi-1 and the Sox9 transcription factor. Normal mouse colon epithelium lacks Paneth cells, a key small intestine niche cell type, and Paneth cell differentiation is dependent on Sox9 function. In Apc-deficient colon epithelium, ectopic Paneth-like cells were seen outside the crypt base, such as new crypt budding sites. Our data indicate Apc inactivation via CDX2P-CreER(T2) targeting in mouse colon epithelium is sufficient to induce adenomatous changes and the generation of Paneth-like cells from neoplastic progenitors, with potentially significant roles in colon adenoma development and progression.

Cre-based deletion of COX-2 within B cells in vitro: steps toward specific nanoparticle-driven COX-2 inactivation in vivo. (P4044)

Abstract Several lines of evidence indicate that COX-2 and downstream prostaglandin (PG) positively influence germinal center development and Ig heavy chain class switching. Although activated B cells upregulate COX-2 and downstream effector molecules, it is unclear whether autocrine production of PG is a major driver of B cell autoimmunity. One approach to investigate this is through Cre-mediated, B cell lineage-specific COX-2flox recombination in mice prone to autoimmunity. Because Cre insertion into B lineage genes, CD19 and mb-1, compromises expression of molecules needed for BCR-triggered autoimmunity, we are seeking to achieve B cell-specific Cre-Lox recombination through an alternative route: B cell-specific NP bearing Cre as a payload. We here report that purified recombinant Cre protein rapidly achieves specific in vitro recombination within PCR amplicons of COX-2 with inserted pLox sites. Furthermore, one hour exposure of viable splenocytes from NOD.B10 COX-2flox mice to Cre protein at 37oC results in ~ 90% recombination. Cells treated with active Cre and subsequently stimulated with BCR-L, IL-4 and BAFF, display significantly less COX-2 protein upon reculture than cells similarly treated with heat-inactivated Cre. We additionally report the preparation of CD22-specific nanoparticles composed of PEG and PLA. In future studies, similar NP will be employed to target Cre specifically to B cells in Sjogren’s Syndrome-susceptible NOD.B10 COX-2flox mice.

Relative antioxidant activities of quercetin and its structurally related substances and their effects on NF-κB/CRE/AP-1 signaling in murine macrophages.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by the oxidative burst in activated macrophages and neutrophils cause oxidative stress-implicated diseases. Quercetin is flavonoid that occurs naturally in plants and is widely used as a nutritional supplement due to its antioxidant and anti-inflammatory properties. In this study, we investigated antioxidant activities and mechanisms of action in zymosan-induced macrophages of quercetin and quercetin-related flavonoids such as quercitrin, isoquercitrin, quercetin 3-O-β-(2″-galloyl)-rhamnopyranoside (QGR) and quercetin 3-O-β-(2″-galloyl)-glucopyranoside (QGG) as well as gallic acid, a building moiety of QGR and QGG. QGR and QGG exhibited stronger antioxidant activities compared with quercetin, whereas quercitrin, isoquercitrin and gallic acid exhibited weak-to-no antioxidant activities, assessed by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging, superoxide production, superoxide scavenging, nitric oxide (NO) production, peroxynitrite (ONOO−) scavenging and myeloperoxidase (MPO) activity. Regarding mechanisms, the quercetin-containing flavonoids QGR and QGG differentially targeted compared with quercetin in the NF-κB signaling pathway that inhibited the DNA binding activity of the NF-κB complex without affecting the degradation and phosphorylation of IκBα and NF-κB phosphorylation. In addition, QGR and QGG inhibited CRE and activator protein (AP-1) transcriptional activity and JNK phosphorylation by inhibiting the cAMP/protein kinase A (PKA) and protein kinase C (PKC) signaling in a different manner than quercetin. Our results showed that although QGR and QGG exhibited stronger antioxidant activities than quercetin in macrophages, their mechanisms of action in terms of the NF-κB, PKA and PKC signaling pathways were different.

A Site-Specific Recombinase-Based Method to Produce Antibiotic Selectable Marker Free Transgenic Cattle

Antibiotic selectable marker genes have been widely used to generate transgenic animals. Once transgenic animals have been obtained, the selectable marker is no longer necessary but raises public concerns regarding biological safety. The aim of this study was to prepare competent antibiotic selectable marker free transgenic cells for somatic cell nuclear transfer (SCNT). PhiC31 intergrase was used to insert a transgene cassette into a “safe harbor” in the bovine genome. Then, Cre recombinase was employed to excise the selectable marker under the monitoring of a fluorescent double reporter. By visually tracking the phenotypic switch from red to green fluorescence, antibiotic selectable marker free cells were easily detected and sorted by fluorescence-activated cell sorting. For safety, we used phiC31 mRNA and cell-permeant Cre protein in this study. When used as donor nuclei for SCNT, these safe harbor integrated marker-free transgenic cells supported a similar developmental competence of SCNT embryos compared with that of non-transgenic cells. After embryo transfer, antibiotic selectable marker free transgenic cattle were generated and anti-bacterial recombinant human β-defensin-3 in milk was detected during their lactation period. Thus, this approach offers a rapid and safe alternative to produce antibiotic selectable marker free transgenic farm animals, thereby making it a valuable tool to promote the healthy development and welfare of transgenic farm animals.

Improvement of pronuclear injection-based targeted transgenesis (PITT) by iCre mRNA-mediated site-specific recombination

We recently reported a novel transgenic (Tg) technique named pronuclear injection-based targeted transgenesis (PITT) (Ohtsuka et al. 2010; Ohtsuka et al. 2012a). The PITT technique enables targeted integration of a single-copy transgene into a specified locus by Cre-loxP mediated recombination and it guarantees faithful and reproducible transgene expression. As described in the first report, the PITT technique requires co-injection of a Cre expression plasmid and a donor vector containing loxP sequences into pronuclei of zygotes obtained from the seed mice that carry loxP sequences at the Rosa26 or H2-Tw3 loci. This method has a (targeted) integration efficiency of 26/585 (4.4 %; 95 % CI 2.9–6.4 %) (Ohtsuka et al. 2012b). Although PITT has a lower transgenic rate (4.4 %) when compared to traditional transgenesis (*10–20 % (Fielder and Montoliu 2011)), it is superior to the latter method since just obtaining one transgenic founder is sufficient in case of PITT. With an aim to improve the PITT technique further we attempted to co-inject iCre mRNA [codon improved Cre recombinase; (Shimshek et al. 2002)] into zygotes with the expectation that the Cre protein would be available more readily in the zygotes from injected iCre mRNA compared to when the Creexpression plasmid DNA was co-injected. We coinjected 10 ng/ll of pAOM donor vector (Ohtsuka et al. 2010) along with various concentrations of iCre mRNA (1–45 ng/ll) into zygotes obtained from the Electronic supplementary material The online version of this article (doi:10.1007/s11248-013-9703-x) contains supplementary material, which is available to authorized users.

Lcn5 Promoter Directs the Region-Specific Expression of Cre Recombinase in Caput Epididymidis of Transgenic Mice1

Epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. To study the function of genes in the caput epididymidis using the Cre/loxP system, we generated Lcn5-Cre transgenic mice in which the expression of Cre recombinase is driven by the 1.8-kb Lcn5 promoter. A total of 11 founder mice carrying the Lcn5-Cre transgene were identified by PCR from 38 offspring, and the integration efficiency was 28.9%. However, only 1 of the 11 transgenic mouse lines were revealed with the Cre recombinase expressed specifically in caput epididymidis. Furthermore, expression of Cre mRNA was first observed on Postnatal Day 30 and continued to increase during development. Subsequently, Cre protein distribution was assessed by crossing Lcn5-Cre transgenic mice into the mT/mG reporter line. As expected, the Cre recombinase activity was only found in principal cells of the middle/distal caput epididymidis. The tissue-specific expression of Cre protein in the caput epididymidis was further confirmed using Lcn5-Cre mice crossed with a mouse strain carrying Aip1 conditional alleles (Aip1(flox/+)). In summary, a transgenic mouse line expressing Cre recombinase in principal cells of caput epididymidis was established. This transgenic mouse line can be used to generate conditional, caput epididymidis-specific knockout mouse models by crossing with mice harboring floxed (LoxP flanked) genes.