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.

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.

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.

Polymorphisms in the human apolipoprotein E receptor 2 gene in Japanese sporadic Alzheimer's disease patients.

Apolipoprotein E (apoE) polymorphism is associated with onset of Alzheimer's disease (AD). We found seven polymorphisms in apoE receptor 2 (ApoER2), an apoE-binding receptor, in Japanese sporadic AD patients, but no association of ApoER2 polymorphisms with AD. We consider that the functions of ApoER2 in the brain may be compensated for by those of other apoE-binding receptors such as VLDL receptor.

Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERß, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

Availability of empty zona pellucida for generating embryonic chimeras.

In the present study we used an empty zona pellucida derived from hatched blastocysts as an alternative source for embryo aggregation and compared results with the conventional microwell method. Denuded 4-cell stage porcine embryos were aggregated by introduction into an empty zona or placement within a concave microwell. The present study showed that although the rate of aggregate formation was similar, the blastocyst rates and allocation of more cells to the inner cell mass (ICM) in the resultant aggregates were increased significantly more in the empty zona than in the microwell. Notably, using an empty zona showed no limitations with regards to the increased number of embryos aggregated or embryonic stages for aggregation, while partial or no aggregation frequently occurred in the microwell. The discrepancy may be due to the difference of microenvironments where the embryos were placed namely, the presence/absence of zona pellucida. We hypothesize the success of the empty zona in generating aggregates is due to the physical aggregation of individual embryos allowing closer contact between the blastomeres and/or embryos compared with a concave microwell. These results indicate that aggregation conditions could influence overall production efficiency and developmental potential of aggregates, suggesting physical restraint via empty zona that provide three-dimensional pressures is an important factor for successful embryo aggregation.

Amino acid supplementation affects imprinted gene transcription patterns in parthenogenetic porcine blastocysts.

To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos.

The effects of personal resources and coping strategies on depression and anxiety in patients with chronic obstructive pulmonary disease.

OBJECTIVES: Little is known about how personal resources and coping strategies influence depression and anxiety in patients with COPD. The purpose of this study was to examine the effect of personal resources on the coping strategies and psychological responses and the mediating role of coping strategies on the relationship of personal resources to depression and anxiety in patients with COPD using a path model. METHODS: This was a cross-sectional correlational study. The participants were 209 patients diagnosed with COPD in South Korea. Personal resources were defined as knowledge of COPD, self-efficacy, and social support. Coping includes both problem-oriented and affect-oriented coping strategies. RESULTS: Higher levels of knowledge of the disease were associated with fewer depressive symptoms; and this relationship was partially mediated by problem-oriented coping strategies. Higher levels of self-efficacy were related to less depression and anxiety. Patients with more perceived social support experienced fewer depressive symptoms through the use of problem-oriented coping strategies. CONCLUSIONS: Our findings suggest that problem-oriented coping strategies may explain the mechanism of how knowledge of the disease and social support influence depressive symptoms for patients with COPD. Further interventions focusing on modifiable factors, including personal resources and problem-oriented coping strategies, are warranted to improve psychological outcomes for patients with COPD.

Generation of porcine fibroblasts overexpressing 11ß-HSD1 with adipose tissue-specific aP2 promoter as a porcine model of metabolic syndrome.

Metabolic syndrome arises from a combination of disorders that increase the risk of cardiovascular disease and diabetes. In previous studies, it was observed that overexpression of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) induced obesity and the insulin resistance that accompanies metabolic syndrome in rodent adipose tissue. Based on these observations, it was hypothesized that overexpression of 11ß-HSD1 may be suitable for the generation of a porcine model of metabolic syndrome. It was evaluated that promoter activities of the porcine adipose fatty acid-binding protein (aP2) gene generates adipose tissue-specific 11ß-HSD1 expression. In adipose tissue, the maximum promoter activity (-2,826 to +51 nt) of aP2 was 200-fold higher than that of a promoterless construct. In addition, 11ß-HSD1 transcriptional levels were significantly increased following the introduction of the aP2 promoter into 3T3­L1 adipocytes. These observations indicate that the aP2 promoter may facilitate 11ß-HSD1 overexpression in porcine adipose tissue. Transgenic fibroblasts were generated containing 11ß-HSD1 cDNA controlled by the aP2 promoter with two screening markers, green fluorescence protein and a neomycin-resistance gene. It was hypothesized that transgenic fibroblasts may be useful for generating a porcine model of metabolic syndrome.

Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes.

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

Proteomic Analysis of Cellular and Membrane Proteins in Fluconazole-Resistant Candida glabrata.

OBJECTIVES: Candida glabrata is one of the most common causes of Candida bloodstream infections worldwide. Some isolates of C glabrata may be intermediately resistant to azoles, with some strains developing resistance during therapy or prophylaxis with fluconazole. In this study, we used a proteomic approach to identify differentially expressed proteins between fluconazoleresistant and -susceptible strains. METHODS: Membrane and cellular proteins were extracted from fluconazolesusceptible and fluconazole-resistant C glabrata strains. Differentially expressed proteins were compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with >1.5-fold difference in expression were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 65 proteins were differentially expressed in the cellular and membrane fractions. Among the 39 cellular proteins, 11 were upregulated and 28 were downregulated in fluconazole-resistant strains in comparison with fluconazole-susceptible strains. In the membrane fraction, a total of 26 proteins were found, of which 19 were upregulated and seven were downregulated. A total of 31 proteins were identified by LC-MS/MS that are involved in glycolysis, carbohydrate transport, energy transfer, and other metabolic pathways. Heat shock proteins were identified in various spots. CONCLUSION: Heat shock and stress response proteins were upregulated in the membrane fraction of the fluconazole-resistant C glabrata strain. Compared with susceptible strains, fluconazole-resistant strains showed increased expression of membrane proteins and decreased expression of cellular proteins.

X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.