Histone deacetylases inhibitor and RAD51 recombinase increase transcription activator-like effector nucleases-mediated homologous recombination on the bovine ß-casein gene locus.

OBJECTIVE: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the ß-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. METHODS: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. RESULTS: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. CONCLUSION: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.

Melatonin improves the meiotic maturation of porcine oocytes by reducing endoplasmic reticulum stress during in vitro maturation.

Under endoplasmic reticulum (ER)-stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER-stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER-stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus-oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription-polymerase chain reaction analysis. Treatment with the ER-stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm-treated groups (1, 5, and 10 µg/mL). We confirmed the reducing effects of melatonin (0.1 µmol/L) on ER-stress after pretreatment with Tm (5 µg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 µmol/L) to Tm-pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER-stress during IVM of porcine oocytes.

Native plants (Phellodendron amurense and Humulus japonicus) extracts act as antioxidants to support developmental competence of bovine blastocysts.

OBJECTIVE: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. METHODS: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 µg/mL) and H. japonicus (0.01 µg/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. RESULTS: We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense (28.9%±2.9%), H. japonicus (30.9%±1.5%), and a mixture of P. amurense and H. japonicus (34.8%± 2.1%) treated groups compared with the control group (25.4%±1.6%). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. CONCLUSION: These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine ß-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine ß-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the ß-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine ß-casein gene.

Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination.

The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using 300 µg/mL G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene.

Hippocampal dysfunctions in tumor-bearing mice.

Individuals with cancer are particularly susceptible to depression and cognitive impairment. However, the precise mechanisms underlying cancer-induced hippocampal dysfunction are poorly understood. We investigated the effects of a peripheral tumor on emotional behavior, hippocampus-dependent memory and associated molecular and cellular features using an experimental animal model. Behavioral alterations were examined; stress-related parameters measured; hippocampal neurogenesis evaluated; and the levels of pro-inflammatory cytokines, brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (COX-2) assayed, 2 weeks after inoculation of adult BALB/c mice with cells of a colon carcinoma cell line (CT26). As the tumors developed, CT26-inoculated mice showed significant increases in the depression-like behavior (measured using the tail suspension test) and memory impairment (in terms of object recognition) compared with vehicle-inoculated controls. The presence of a peripheral tumor significantly elevated the hippocampal levels of mRNAs encoding interleukin-6 (IL-6) and tumor necrosis factor-α, as well as plasma IL-6 and corticosterone levels. Additionally, the adrenal glands became enlarged, and the numbers of Ki-67-positive proliferating hippocampal cells and doublecortin-positive immature progenitor neurons, as well as the constitutive levels of mRNAs encoding BDNF and COX-2 were significantly reduced. Therefore, a peripheral tumor alone may be sufficient to induce hippocampal dysfunction, possibly by reducing the rate of neurogenesis and the levels of BDNF and COX-2 in that tissue and also by increasing stress-related parameters and the circulating levels of pro-inflammatory cytokines.

Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into ß-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein.

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ß-casein gene locus using a knock-in vector for the ß-casein gene locus. We developed the knock-in vector on the porcine ß-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ß-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ß-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ß-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

Disruption of early embryonic development in mice by polymethylmethacrylate nanoplastics in an oxidative stress mechanism.

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 µg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 µg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 µg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 µg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

Epigenetic signatures and temporal expression of lineage-specific genes in hESCs during differentiation to hepatocytes in vitro.

Embryonic stem cells (ESCs) maintain unique epigenetic states to maintain their pluripotency. Differentiation of ESCs into specialized cell types requires changes in these epigenetic states. However, the dynamics of epigenetic marks found in hESCs during differentiation are poorly understood. Here, we report the variation in the dynamics of epigenetic modifications associated with the expression of lineage-specific genes during differentiation of hESCs to hepatocytes in vitro. The promoter regions of pluripotency marker genes characterized by permissive histone marks such as trimethylation of H3 at lysine 4 (H3K4me3) and acetylation of H3 at lysine 9 (H3K9ac) in hESCs were instead enriched with repressive histone marks such as dimethylation of H3 at lysine 9 (H3K9me2), trimethylation of H3 at lysine 9 (H3K9me3) and trimethylation of H3 at lysine 27 (H3K27me3) during differentiation to hepatocytes. Interestingly, expression of definitive endoderm marker genes containing bivalent and non-bivalent domains may be modulated by a marked reduction in H3K27me3 and a significant enhancement of permissive marks such as H3K4me3 and H3K9ac during hESC differentiation. Expression of hepatocyte marker genes regulated by histone modifications was similar to that of pluripotency marker genes. Our findings provide insight into the epigenetic mechanisms regulating expression of developmental genes. Of particular interest, they may be differentially regulated either in a bivalent or non-bivalent domain manner during hESC differentiation.

Nucleofection-mediated α1,3-galactosyltransferase gene inactivation and membrane cofactor protein expression for pig-to-primate xenotransplantation.

Xenotransplantation of pig organs into primates leads to hyperacute rejection (HAR). Functional ablation of the pig α 1,3-galactosyltransferase (GalT) gene, which abrogates expression of the Gal α 1-3Gal ß 1-4GlcNAc-R (Gal) antigen, which inhibits HAR. However, antigens other than Gal may induce immunological rejection by their cognate antibody responses. Ultimately, overexpression of complement regulatory proteins reduces acute humoral rejection by non-Gal antibodies when GalT is ablated. In this study, we developed a vector-based strategy for ablation of GalT function and concurrent expression of membrane cofactor protein (MCP, CD46). We constructed an MCP expression cassette (designated as MCP-IRESneo) and inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Nucleofection of porcine ear skin fibroblasts using the U-023 and V-013 programs resulted in high transfection efficiency and cell survival. We identified 28 clones in which the MCP-IRESneo vector had been successfully targeted to exon 9 of the GalT gene. Two of those clones, with apparent morphologically mitotic fibroblast features were selected through long-term culture. GalT gene expression was downregulated in these 2 clones. Importantly, MCP was shown to be efficiently expressed at the cell surface and to efficiently protect cell lysis against normal human complement serum attack in vitro.

Enzyme Feed Additive with Arazyme Improve Growth Performance, Meat Quality, and Gut Microbiome of Pigs.

The supplementation of pig diets with exogenous enzymes is widely used with the expectation that it will improve the efficiency of nutrient utilization, thereby, improving growth performance. This study aims to evaluate the effects of a 0.1% (v/v) multi-enzyme (a mixture of arazyme (2,500,000 Unit/kg), xylanase (200,000 Unit/kg) and mannanase (200,000 Unit/kg)) supplementation derived from invertebrate symbiotic bacteria on pig performance. Here, 256 growing pigs were assigned to control and treatment groups, respectively. The treatment group exhibited a significantly reduced average slaughter age; the final body weight and average daily gain increased compared with that of the control group. In the treatment group, the longissimus muscle showed a remarkable decrease in cooking loss, shear force, and color values with increased essential and non-essential amino acid concentrations. Furthermore, the concentrations of mono- and polyunsaturated fatty acids in the treatment group increased. Feed additive supplementation increased the family of Ruminococcaceae and genera Lactobacillus, Limosilactobacillus, Turicibacter, and Oscillibacter, which play a positive role in the host physiology and health. Predicted metabolic pathway analysis confirmed that operational taxonomic units and predicted amino acid biosynthesis pathways were strongly associated. The results suggest that applying exogenous enzymes derived from invertebrate symbiotic bacteria enhances animal performance.

Regulation of OCT4 gene expression by liver receptor homolog-1 in human embryonic carcinoma cells.

We demonstrate the regulation of OCT4 gene expression mediated by liver receptor homolog-1 (LRH-1) in human embryonic carcinoma cells. LRH-1 and OCT4 are co-expressed in undifferentiated NCCIT cells and decreased during retinoic acid-induced differentiation. Dose-dependent overexpression of LRH-1 transactivated the OCT4 promoter activity and its dominant negative form with a deletion of activation function-2 motif reduced the activity even in the presence of LRH-1. The OCT4 promoter contains potent three LRH-1 binding sites; one within conserved region (CR) 1 and two within CR2. Mutagenesis of each binding site affected the decrease in OCT4 promoter activity and the 2nd binding site mutant most significantly reduced the transcriptional activity, compared to that of 1st and 3rd binding site mutants, respectively. Simultaneous disruption of 2nd and 3rd binding sites led to significant down-regulation of the activity even in the presence of 1st binding site-containing CR1. Moreover, mutation of each binding element within native or exogenous minimal promoter-driven CR1 or CR2 also decreased the promoter activity to some different extent, suggesting that three binding elements may be implicated in the induction of the full-activity of OCT4 promoter. In vivo binding assay revealed the 2nd and 3rd binding motifs within CR2 were more enriched than the 1st one within CR1. Taken together, our study indicates that LRH-1 acts as a transcriptional activator in the regulation of OCT4 gene expression through the cooperative interaction with three binding sites directly or/and indirectly.

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus.

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Cloning and Molecular Characterization of Porcine ß-casein Gene (CNS2).

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ß-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ß-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ß-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ß. In addition, the first intron of the porcine ß-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ß, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ß-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ß-casein gene. The ß-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ß-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ß-casein gene activity.

Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine ß-casein gene locus.

OBJECTIVE: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine ß-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). METHODS: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine ß-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). RESULTS: Treatment with CdCl2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. CONCLUSION: Treatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

Arazyme in combination with dietary carbohydrolases influences odor emission and gut microbiome in growing-finishing pigs.

This study evaluated the effects of supplementing feed with arazyme and dietary carbohydrolases derived from invertebrate gut-associated symbionts on the noxious gas emissions, gut microbiota, and host-microbiome interactions of pigs. Here, 270 and 260 growing pigs were assigned to control and treatment groups, respectively. The tested feed additives contained a mixture of arazyme (2,500,000 Unit/kg) and synergetic enzymes, xylanase (200,000 Unit/kg) and mannanase (200,000 Unit/kg), derived from insect gut-associated symbionts in a 7.5:1:1 ratio. The control group was fed a basal diet and the treatment group was fed the basal diet supplemented with 0.1 % enzyme mixture (v/v) for 2 months. Odorous gases were monitored in ventilated air from tested houses. Fecal samples were collected from steel plate under the cage at the completion of the experiment to determine chemical composition, odor emissions, and bacterial communities. There was a significant decrease in the concentration of NH3 (22.5 vs. 11.2 ppm; P < 0.05), H2S (7.35 vs. 3.74 ppm; P < 0.05), trimethylamine (TMA) (0.066 vs. 0.001 ppm; P < 0.05), and p-cresol (0.004 ppm vs. 0 ppm; P < 0.05) at 56 d in treatment group compared with the control group. Moreover, fecal analysis results showed that exogenous enzyme supplementation caused a reduction in VFAs and indole content with approximately >60 % and 72.7 %, respectively. The result of gas emission analysis showed that NH3 (9.9 vs. 5.3 ppm; P < 0.05) and H2S (5.8 vs. 4.1 ppm; P < 0.05) were significantly reduced in the treatment group compared to the control group. The gut microbiota of the treatment group differed significantly from that of the control group, and the treatment group altered predicted metabolic pathways, including sulfur and nitrogen related metabolism, urea degradation. The results demonstrated that supplementing feed with arazyme with dietary carbohydrolases effectively controls noxious gas emissions and improves health and meat quality of pigs.

Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum.

Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken ß-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-ß-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.

Molecular characterization and expression of the low-density lipoprotein receptor-related protein-10, a new member of the LDLR gene family.

We report the characterization of a new member of the low-density lipoprotein receptor (LDLR) gene family designated LRP10. Human LRP10 cDNA encodes a 1905 amino acid type I membrane protein consisting of five functional domains characteristic of the LDLR gene family. CHO-ldlA7 cells transfected with human LRP10 cDNA bound LDLR-associated protein, but not beta-VLDL and HDL. Human LRP10 transcripts were primarily found in the brain, muscle and heart. In situ hybridization of the rat brain showed that the transcripts were intensely present in the cerebral cortex, hippocampus, choroid plexus, ependyma and granular layer. In the developing rat brain, transcript levels gradually increased from postnatal day 1 to 20. Immunofluorescence analysis indicated that LRP10 was observed in the ventricular zone of the embryonic day 14.5 mouse cerebral cortex. The present studies suggest that LRP10 may play a significant role in the brain physiology other than lipoprotein metabolism.

The low-density lipoprotein receptor-related protein 10 is a negative regulator of the canonical Wnt/beta-catenin signaling pathway.

Wnt signaling pathways play fundamental roles in the differentiation, proliferation and functions of many cells as well as developmental, growth, and homeostatic processes in animals. Low-density lipoprotein receptor (LDLR)-related protein (LRP) 5 and LRP6 serve as coreceptors of Wnt proteins together with Frizzled receptors, triggering activation of canonical Wnt/beta-catenin signaling. Here, we found that LRP10, a new member of the LDLR gene family, inhibits the canonical Wnt/beta-catenin signaling pathway. The beta-catenin/T cell factor (TCF) transcriptional activity in HEK293 cells was activated by transfection with Wnt3a or LRP6, which was then inhibited by co-transfection with LRP10. Deletion of the extracellular domain of LRP10 negated its inhibitory effect. The inhibitory effect of LRP10 was consistently conserved in HEK293 cells even when GSK3beta phosphorylation was inhibited by incubation with lithium chloride and co-transfection with constitutively active S33Y-mutated beta-catenin. Nuclear beta-catenin accumulation was unaffected by LRP10. The present studies suggest that LRP10 may interfere with the formation of the beta-catenin/TCF complex and/or its binding to target DNA in the nucleus, and that the extracellular domain of LRP10 is critical for inhibition of the canonical Wnt/beta-catenin signaling pathway.

Pear Extract and Malaxinic Acid Reverse Obesity, Adipose Tissue Inflammation, and Hepatosteatosis in Mice.

SCOPE: Obesity and diabetes are major public health problems and are emerging as pandemics. Considerable evidence suggests that pear fruit consumption is associated with a lower risk of obesity-related complications. Thus, the present study is conducted to investigate the therapeutic potential of pear extract (PE) for reversing obesity and associated metabolic complications in high-fat diet-induced obese mice. METHODS AND RESULTS: Obesity is induced in male C57BL/6 mice fed a high-fat diet for 11 weeks. After the first 6 weeks on the diet, obese mice are administered vehicle or PE for 5 weeks. PE treatment decreases body weight gain, expands white adipose tissue (WAT), and causes hepatic steatosis in obese mice, as well as inhibits adipogenesis and lipogenesis. Impaired glucose tolerance and insulin resistance are improved by PE. In addition, PE reduces macrophage infiltration and expression of pro-inflammatory genes and deactivates mitogen-activated protein kinases in WAT. Finally, malaxinic acid is identified as an active component responsible for the anti-obesity effects of PE in mice. CONCLUSION: The results demonstrate that PE supplementation ameliorates diet-induced obesity and associated metabolic complications and suggest the health-beneficial effects of both pear fruits and malaxinic acid in counteracting these diseases.