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In this study, we aim to investigate the effects of cooling water temperature and deadman status on thermal flow in the hearth of a blast furnace (BF) during tapping shutdown according to the geometric model and boundary conditions given from the BF No.2 that has three asymmetrical tapholes in the hearth of BF at Dragon Steel, Taiwan. The mathematical formula includes the assumption of incompressible fluid, transient turbulent momentum conservation with standard k-emodel, non-Darcy porous media, Boussinesq approximation, transient turbulent energy conservation, and conjugate heat transfer between solid and fluid. It is simulated that the tapping shutdown is induced by No. 2 taphole from tapping to shutdown while No. 1 and No.3 tapholes are always closed. As shown in the numerical results, the wall heat flux near the taphole area increased significantly with the decrease of cooling water temperature. When the cooling water temperature was decreased from 35 °C to 10 °C, the wall heat flux near No.2, No.1, and No.3 tapholes increases by 11.5%, 30.4%, and 30.4% respectively. During tapping shutdown for one hour, the wall heat fluxes near the taphole area for gutter deadman and floating deadman in the blast furnace hearth were increased by 0.94% and 1.46%, respectively. These results would be useful to optimize operating conditions of blast furnace hearth, thus reducing the ironmaking cost.

Chronic obstructive pulmonary disease (COPD) is a sustained blockage of the airways due to lung inflammation occurring with chronic bronchitis and/or emphysema. Progression of emphysema may be slowed by vascular endothelial growth factor A (VEGFA), which reduces apoptotic tissue depletion. Previously, authors of the present report demonstrated that cis-resveratrol (c-RSV)-induced heat-shock protein 70 (HSP70) promoter-regulated VEGFA expression promoted neovascularization of genetically modified mesenchymal stem cells (HSP-VEGFA-MSC) in a mouse model of ischemic disease. Here, this same stem cell line was evaluated for its protective capacity to alleviate elastase-induced pulmonary emphysema in mice. Results of this study showed that c-RSV-treatment of HSP-VEGFA-MSC exhibited synergy between HSP70 transcription activity and induced expression of anti-oxidant-related genes when challenged by cigarette smoke extracts. Eight weeks after jugular vein injection of HSP-VEGFA-MSC into mice with elastase-induced pulmonary emphysema followed by c-RSV treatment to induce transgene expression, significant improvement was observed in respiratory functions. Expression of VEGFA, endogenous nuclear factor erythroid 2-related factor (Nrf 2), and manganese superoxide dismutase (MnSOD) was significantly increased in the lung tissues of the c-RSV-treated mice. Histopathologic examination of treated mice revealed gradual but significant abatement of emphysema and restoration of airspace volume. In conclusion, the present investigation demonstrates that c-RSV-regulated VEGFA expression in HSP-VEGFA-MSC significantly improved the therapeutic effects on the treatment of COPD in the mouse, possibly avoiding side effects associated with constitutive VEGFA expression.

Summary: Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs) have been efficiently achieved by somatic cell nuclear transfer (SCNT). We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/−) mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells. : Sung et al. demonstrate in a mouse model that telomeres of telomerase haplo-insufficient cells can be elongated by somatic cell nuclear transfer. Moreover, ntESCs derived from Terc+/− cells exhibit pluripotency evidenced by generation of Terc+/−ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency.

The ability of adult stem cells to reside in a quiescent state is crucial for preventing premature exhaustion of the stem cell pool. However, the intrinsic epigenetic factors that regulate spermatogonial stem cell quiescence are largely unknown. Here, we investigate in mice how DNA methyltransferase 3-like (DNMT3L), an epigenetic regulator important for interpreting chromatin context and facilitating de novo DNA methylation, sustains the long-term male germ cell pool. We demonstrated that stem cell-enriched THY1+ spermatogonial stem/progenitor cells (SPCs) constituted a DNMT3L-expressing population in postnatal testes. DNMT3L influenced the stability of promyelocytic leukemia zinc finger (PLZF), potentially by downregulating Cdk2/CDK2 expression, which sequestered CDK2-mediated PLZF degradation. Reduced PLZF in Dnmt3l KO THY1+ cells released its antagonist, Sal-like protein 4A (SALL4A), which is associated with overactivated ERK and AKT signaling cascades. Furthermore, DNMT3L was required to suppress the cell proliferation-promoting factor SALL4B in THY1+ SPCs and to prevent premature stem cell exhaustion. Our results indicate that DNMT3L is required to delicately balance the cycling and quiescence of SPCs. These findings reveal a novel role for DNMT3L in modulating postnatal SPC cell fate decisions.

Liver diseases, which can be caused by alcohol abuse, chemical intoxication, viral hepatitis infection, and autoimmune disorders, are a significant health issue because they can develop into liver fibrosis and cirrhosis. Lactoferrin (LF), a siderophilic protein with 2 iron-binding sites, has been demonstrated to possess a multitude of biological functions, including antiinflammation, anticancer, and antimicrobial effects, as well as immunomodulatory-enhancing functions. In the current study, we induced hepatotoxicity in rats with dimethylnitrosamine (DMN) to establish a situation that would enable us to evaluate the hepatoprotective effects of LF against hepatic injury. Our results showed that DMN-induced hepatic pathological damage significantly decreased the body weight and liver index, increased the mRNA and protein levels of collagen α-1(I) (ColIα-1) and α-smooth muscle actin, and increased the hydroxyproline content. However, treatment with LF significantly increased body weight and liver index, decreased the mRNA and protein levels of ColIα-1 and α-smooth muscle actin, and suppressed the hydroxyproline content when compared with the DMN-treated group. Liver histopathology also showed that low-dose LF (100mg/kg of body weight) or high-dose LF (300 mg/kg of body weight) could significantly reduce the incidences of liver lesions induced by DMN. These results suggest that the LF exhibits potent hepatoprotection against DMN-induced liver damage in rats and that the hepatoprotective effects of LF may be due to the inhibition of collagen production and to stellate cell activation.

Estrogen and progestins have adverse effects, and many of these adverse effects are caused by progestins. Due to this, many women choose to use botanical alternatives for hormone replacement therapy, which does not trigger steroidogenic properties. Therefore, it is necessary to screen these herbs for progestogenic and anti-progestogenic properties. Extract of 13 Chinese medicinal plants were analysed for progestogenic and anti-progestogenic activities by using progesterone response element-driven luciferase reporter gene bioassay. MTT assay was carried out to investigate the cytotoxic effect of herb extract on PAE cells. Among the 13 herbs, Dipsacus asperoides extract exhibited progestogenic activity, and 10 species - Cortex eucommiae, Folium artemisiae argyi, Glycyrrhiza uralensis, Angelica sinensis, Atractylodes macrocephala koidz, Scutellaria baicalensis, Cuscuta chinensis, Euscaphis japonica, Ailanthus altissima, and Dioscorea opposita - were recognized to have anti-progestogenic like activities. Extract of Dipsacus asperoides demonstrated dose-dependent progestogenic activity, and the progestogenic activity of 100 (mu)g/mL extracts was equivalent to 31.45 ng/mL progesterone activity. Herbs extracts that exhibited anti-progestogenic-like activity also inhibited the 314.46 ng/mL progesterone activity in a dose-response manner. None of the herb extracts shown significant toxic effect on PAE cells at 40-100 (mu)g/mL compared to control. This discovery will aid selection of suitable herbs for hormone replacement therapy.

Survival motor neuron (SMN) is the determining factor in spinal muscular atrophy, the most common genetic cause of childhood mortality. We have previously found that SMN regulates stem cell division, proliferation and differentiation in Drosophila. However, it is unknown whether a similar effect exists in vertebrates. Here, we show that SMN is enriched in highly proliferative embryonic stem cells (ESCs) in mice and reduction of SMN impairs the pluripotency of ESCs. Moreover, we find that SMN reduction activates ERK signaling and affects neuronal differentiation in vitro. Teratomas with reduced SMN grow more slowly and show weaker signals of neuronal differentiation than those with a normal level of SMN. Finally, we show that over-expression of SMN is protective for ESCs from retinoic acid-induced differentiation. Taken together, our results suggest that SMN plays a role in the maintenance of pluripotent ESCs and neuronal differentiation in mice.

In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygotes. These reprogramming systems play crucial roles in regulating genome functions during critical stages of embryonic development, and they confer the stability of gene expression during mammalian development. The frequent unexpected loss of progeny from somatic cell nuclear transfer (SCNT) is an ongoing problem. In this study, we used six cloned bovines (named NT-1 to NT-6), which were created by ear fibroblast nuclear transfer and displayed short life spans with multiple organ defects, as an experimental model. We focus here on three imprinted genes (IGF2, H19, and XIST) and four satellite loci (Satellite I, Satellite II, Art2, and VNTR) to investigate their methylation changes. The results revealed that aberrant methylation frequently occurred in the analyzed imprinted genes, but not in the satellite loci, of the cloned bovines. After the bovine fibroblast cells were treated with the 5-aza-2(′)-deoxycytidine (5-Aza-dc) demethylation agent, the methylation percentages of the XIST and H19 putative differentially methylated region (DMR) were significantly decreased (XIST, p

The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P < 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits.

Adiponectin and its receptors have been demonstrated to play important roles in regulating glucose and lipid metabolism in mice. Obesity, type II diabetes and cardiovascular disease are highly correlated with down-regulated adiponectin signaling. In this study, we generated mice overexpressing the porcine Adipor1 transgene (pAdipor1) to study its beneficial effects in metabolic syndromes as expressed in diet-induced obesity, hepatosteatosis and insulin resistance. Wild-type (WT) and pAdipor1 transgenic mice were fed ad libitum with a standard chow diet (Chow) or a high-fat/sucrose diet (HFSD) for 24 weeks, beginning at 6 to 7 weeks of age. There were 12 mice per genetic/diet/sex group. When challenged with HFSD to induce obesity, the pAdipor1 transgenic mice resisted development of weight gain, hepatosteatosis and insulin resistance. These mice had lowered plasma adiponectin, triglyceride and glycerol concentrations compared to WT mice. Moreover, we found that (indicated by mRNA levels) fatty acid oxidation was enhanced in skeletal muscle and adipose tissue, and liver lipogenesis was inhibited. The pAdipor1 transgene also restored HFSD-reduced phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose transporter 4 mRNA in the adipose tissues, implying that the increased Pck1 may promote glyceroneogenesis to reduce glucose intolerance and thus activate the flux of glyceride-glycerol to resist diet-induced weight gain in the adipose tissues. Taken together, we demonstrated that pAdipor1 can prevent diet-induced weight gain and insulin resistance. Our findings may provide potential therapeutic strategies for treating metabolic syndromes and obesity, such as treatment with an ADIPOR1 agonist or activation of Adipor1 downstream targets.

Chemotherapy (CTx)-induced premature ovarian failure (POF) in woman remains clinically irreversible. Amniotic fluid stem cells (AFSCs) have shown the potential to treat CTx-induced POF; however, the underlying mechanism is unclear. Here we demonstrate that AFSC-derived exosomes recapitulate the anti-apoptotic effect of AFSCs on CTx-damaged granulosa cells (GCs), which are vital for the growth of ovarian follicles. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-treated mice via the delivery of microRNAs in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. The down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro. Further, the administration of these miRNAs recapitulates the effects both in vitro and in vivo, in which miR-10a contributes a dominant influence. Our findings illustrate that miR-10a has potential as a novel therapeutic agent for the treatment of POF.

Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB. Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.

Effects of enucleation timing on enucleation rates, development and methylation levels of reconstructed bovine embryos were investigated. However, the enucleation rate of reconstructed embryos produced by the enucleation before fusion and activation (EBFA) was higher than that by the enucleation after fusion and activation (EAFA) procedure (80.7% vs. 59.1%, P0.05). The blastocyst rate of reconstructed embryos cloned with ear fibroblasts in EBFA group was reduced (P0.05) in comparison with that of EAFA group (24.6% vs. 34.4%). Two out of 11 recipients were pregnant and gave birth to two viable calves after transfer of 20 reconstructed EBFA embryos. Two out of seven recipients were pregnant and also gave birth to two calves, with one surviving, after transfer of 12 reconstructed embryos produced by EAFA procedure. Finally, the methylation level of satellite I gene of donor cells (69.8%) and reconstructed embryos in EBFA group (64.7%) were similar, which were both higher (P0.05) than that of the reconstructed embryos in EAFA group (44.4%). The methylation level of satellite I gene of the reconstructed embryos in the IVF embryos (31.9%) was lower (P0.05) than those in all other treatments. In conclusion, the reconstructed bovine embryos produced by the EAFA procedure revealed a better developmental competence with a lower methylation rate of satellite I gene than those produced by the EBFA procedure.

This study was conducted to determine the effect of rabbit oocytes collected from ovaries or oviducts on the developmental potential of nuclear transplant embryos. Donor nuclei were obtained from adult skin fibroblasts, cumulus cells, and embryonic blastomeres. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12 h post-hCG injection. The majority of collected oocytes were still attached to the sites of ovulation on the ovaries. We found that follicular oocytes had a significantly higher rate of fusion with nuclear donor cells than oviductal oocytes. There was no difference in the cleavage rate between follicular and oviductal groups, but morula and blastocyst development was significantly higher in the follicular group than in the oviductal group. Two live clones were produced in follicular group using blastomere and cumulus nuclear donors, whereas one live clone was produced in the oviductal group using a cumulus nuclear donor. These results demonstrate that cloned rabbit embryos derived from follicular oocytes have better developmental competence than those derived from oviductal oocytes.

The aim of this article is to demonstrate and characterize caprine mammary epithelial cells (CMC) immortalized with human telomerase reverse transcriptase (hTERT) gene. Five immortalized CMCs were assigned to either myoepithelial or luminal epithelial groups based on their morphology and expression of cell lineage-specific intermediate filaments. Telomeric repeat amplification protocol revealed various telomerase activities in CMCs associated with their distinct proliferation potential. Karyotypic analysis showed three CMCs retained their modal Capra hircus chromosome number (2n = 60), whereas the remaining two CMCs were abnormal at 2n = 19 and 2n = 36. CMCs with abnormal karyotypes lost p53 protein after chemical-induced DNA damage and showed anchorage-independent growth in soft agar assay. In terms of functional differentiation, luminal CMCs organized into alveolus-like structures when grown in Matrigel. Furthermore, αs1- and β-casein gene was induced in luminal CMCs in response to lacto-hormones stimulation. Together these results showed that hTERT-immortalized CMCs retained major characteristics of mammary epithelial cells, and stability of the genome is required for maintaining normal mammary epithelium function. Application of CMCs can provide valuable models to study alveologenesis and lactogenesis of mammary epithelium and test the feasibility of recombinant constructs designed for the generation of transgenic livestock.

The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

The use of evaporative cooling for mitigating heat stress in lactating cows in humid areas is controversial. In Taiwan, Holstein cow performance is significantly restricted by hot and humid weather. This study investigated the efficacy of using a tunnel-ventilated, water-padded freestall (TP) barn for reducing heat stress in lactating cows. From August to October 2006, 36 cows allocated in a 3×3 Latin square were raised in 3 barn cooling treatments: a conventional freestall barn with fans and sprinklers in the feed line (Fan+SP, control), a TP barn, and a TP barn with sprinkler cooling (TP+SP). Daytime air speeds in the 3 barns were 1.23, 2.38, and 2.06 m/s, respectively. Both TP barns were more efficient than the control in reducing the daytime temperature and temperature-humidity index. The barn temperature was26°C for an extra 4.2h per day, but the relative humidity was96% in both TP barns. Cows in both TP barns had higher respiration rates and skin temperatures at 0300 h than cows in the Fan+SP barn. The TP environment increased the cows' serum cholesterol level and the activities of alkaline phosphatase and alanine aminotransferase, but blood partial pressure of CO(2) was not affected. Vaginal temperature was persistently high in cows in the TP barn; in the 2 SP barns, it decreased 0.4 to 0.6°C following sprinkling and milking. The intake activity and rumen digestion of cows raised in the 3 environments were similar. Cows in both TP barns ingested more dry matter. Cows in the TP+SP barn tended to produce more milk than those in the Fan+SP barn (25.4 vs. 24.7 kg). Although heat stress was not completely alleviated in these 3 barns, the TP+SP treatment resolved the negative effect of a previous TP barn built in 2004 on intake and milk yield by increasing air speed and using sprinkler cooling. Thus, it is expected that TP+SP barns will be beneficial in regions with high humidity. Adequate air speed and sprinkler cooling are likely to be key factors for further study.

Adiponectin (APN), a circulating adipose-derived hormone that regulates inflammation and energy metabolism, has beneficial effects on the cardiovascular disorders. Serum APN levels are lower in patients with coronary artery disease and higher in patients with chronic kidney disease. However, the precise role of APN in acute reno-vascular disease is not clear. Results of the present study show that serum APN concentration decreased after renal ischemia reperfusion (I/R) injury in mice. In addition, I/R-induced renal dysfunction (elevated serum creatinine and urea levels), inflammation (number of infiltrating neutrophils, myeloperoxidase activity), and apoptotic responses (apoptotic cell number and caspase-3 activation) were attenuated in APN-treated compared to control mice. Molecular and biochemical analysis revealed that APN up-regulates heme oxygenase-1 (HO-1) via peroxisome-proliferator-activated-receptor-α (PPARα) dependent pathway which is mediated through the enhancement of COX-2 and 6-keto PGF1α expression. Chromatin immune-precipitation assay demonstrated that APN increases the binding activity of PPARα to PPRE region of HO-1 promoter. Furthermore, APN induced HO-1 expression was only found in wild-type but not in PPARα gene deleted mice. This provides in vivo evidence that APN mediated HO-1 expression depends on PPARα regulation. In conclusion, our results provide a novel APN mediated prostacyclin-PPARα-HO-1 signaling pathway in protecting renal I/R injury.

Amelogenin (AMEL) is a conserved gene located on the sex chromosomes of mammals. It is involved in the formation of enamel, which is the hard, white material that forms the protective outer layer of each tooth. In this study, we first cloned and determined the intron sequences of the goat AMELX and AMELY genes from female and male ear tissues. The polymorphic AMEL alleles were further analyzed by PCR-based RFLP and Southern blot hybridization analyses. Results showed that intron 5 nucleotide sequences of the goat AMELY gene contains multiple deletions/insertions and shares only 48.5% identity to intron 5 of the goat AMELX gene. Based on the polymorphic AMEL intron sequences, a set of sex-specific triplex primers was designed to PCR amplify a single fragment of 264 bp from the X chromosome of female goats and 2 fragments of 264 and 206 bp from the X and Y chromosomes, respectively, of male goats. An increased sensitivity for sex determination was reached with a single blastomere at the blastula stage isolated from goat embryos. A total of 43 goat embryos were used to estimate a 100% accuracy rate of this method confirmed by chromosomal karyotyping and live births. The embryo sexing technique has been successfully applied in different strains of goats including Alpine, Saanen, Nubian, and Taiwan goats.

Visfatin is a visceral adipose tissue-specific adipocytokine that plays a positive role in attenuating insulin resistance by binding to the insulin receptor. Visfatin has been suggested to play a role in the regulation of lipid metabolism and inflammation; however, the mechanism remains unclear. We investigated the effects of visfatin on the regulation of gene expression in cultured porcine preadipocytes and differentiated adipocytes. In preadipocytes, the mRNA abundance of lipoprotein lipase and PPARgamma were significantly increased by visfatin or insulin treatment after 8 d (all P < 0.05). In the presence of insulin, the mRNA abundance of adipocyte fatty acid-binding protein was 24.7-fold greater than in the untreated group (P < 0.05), whereas visfatin alone had no effect on adipocyte fatty acid-binding protein mRNA abundance. Adipocyte differentiation was induced by insulin treatment for 8 d. In differentiated porcine adipocytes, exposure to insulin or visfatin for 24 h increased (P < 0.05) fatty acid synthase mRNA abundance but had no effect on the expression of sterol regulatory element binding-protein 1c mRNA. We also found a 5.8-fold upregulation of IL-6 expression in porcine adipocytes after 24 h of treatment with visfatin (P < 0.05). These results demonstrated that visfatin upregulated lipoprotein lipase expression in preadipocytes, potentially facilitating lipid uptake, and increased the gene expression of fatty acid synthase in differentiated adipocytes to potentially enhance lipogenic activity. Furthermore, visfatin can upregulate IL-6 expression in differentiated porcine adipocytes. The information presented in this study provides insights into the roles of visfatin in lipid metabolism in pigs.

Peroxisome proliferator-activated receptor delta promotes fatty acid catabolism and energy expenditure in skeletal muscle and adipose tissues. A ligand for PPARdelta is required to activate PPARdelta function. Polyunsaturated fatty acids are potential ligands for PPARdelta activation. The current experiment was designed to determine the potential for PUFA, particularly from dietary fish oil, to activate porcine PPARdelta in vivo. Transgenic mice were generated to overexpress porcine PPARdelta in the adipose tissue. Mice were fed a high-saturated fat (13% beef tallow), or high-unsaturated fat (13% fish oil) diet, or a diet containing 4 mg/kg of a PPARdelta ligand (L165041) for 4 mo. Compared with beef tallow feeding, fish oil feeding reduced fat mass and decreased (P < 0.05) plasma triacylglycerol and FFA concentrations in the transgenic mice. Adipose tissue expression of genes involved in adipogenesis (i.e., lipoprotein lipase and adipocyte fatty acid-binding protein) was decreased in transgenic mice fed fish oil or the PPARdelta ligand. In the same mice, expression of the lipolytic gene, hormone-sensitive lipase was increased (P < 0.05). Fish oil feeding also stimulated expression of genes participating in fatty acid oxidation in the liver of transgenic mice compared with wild-type mice. Overall, these results indicate that PUFA may serve as natural and effective regulators of lipid catabolism in vivo and many of these effects may be generated from activation of PPARdelta.

Objectives: Isolation of mouse mesenchymal stem cells (mMSCs), by the approach of plastic adherence, has been difficult due to persistent contamination by haematopoietic cells (HCs); we have observed that this contamination was due to engagement between HCs and mMSCs. The HCs can be lifted together with the mMSCs despite their insensitivity to trypsin digestion. Herein, we provide a single-step procedure to rapidly segregate mMSCs from HC contaminants using transient lower-density plastic adherence (tLDA). Materials and methods: The tLDA was performed by replating bone marrow adherent cells at lower density (1.25 × 104 cells/cm2) than usual, allowing for transient adherence of no more than 3 h, followed by trypsin digestion. tLDA-isolated cells were evaluated by immunophenotyping, multi-differentiation potentials, immunosuppressive properties, and therapeutic potential as demonstrated by symptoms of osteoporosis. Results: The single-step tLDA method can effectively eliminate the persistent HC contaminants; tLDA-isolated cells were phenotypically equivalent to those reported as mMSCs. The isolated cells possessed classic tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages and had immunosuppressive properties. After intravenous transplantation, they migrated into the allogeneic bone marrow and rescued hosts from osteoporosis symptoms, demonstrating their therapeutic potential. Conclusions: We have developed a simple and economical method that effectively isolates HC-free, therapeutically functional mMSCs from bone marrow cell adherent cultures. These cells are suitable for various mechanistic and therapeutic studies in the mouse model.

This study attempted to clarify the difference in eggshell pigmentation between blue-shelled ducks (BSD) and white-shelled ducks (WSD). The eggshell pigmentation deposition process is discussed. Ultraviolet spectro-photometer and HPLC were used to determine the biliverdin concentration in the shell gland, uterus liquid and eggshell at 6, 12, 18, 20, 23.5 h post-oviposition. The biliverdin concentration in the eggshell and uterus fluid showed significant differences between BSD and WSD, but not in the shell gland. The heme oxygenase activity in the shell gland of both kinds of ducks remained mostly constant during the ovulatory cycle with no variation. The assay of exogenous biliverdin injection into the shell gland antrum in the WSD indicated that exogenous biliverdin could be deposited continuously into the eggshell until the source was exhausted. A layer-by-layer dissolution assay was used to examine the eggshell pigment deposition process. The biliverdin concentration in the first to sixth layers of the eggshell in the BSD was significantly higher than that in the white-shelled counterpart. The blue pigment concentration increased persistently from the 6 th layer to the 1 st layer. The BSD eggshells did not accumulate a large quantity of biliverdin in the most external layer. They tended to increase the deposition layer by layer. Our results demonstrated that different BSD and WSD eggshell colors were influenced by the amount of biliverdin in the uterus fluid and not determined by the amount of biliverdin in the shell gland. This implies the existence of a mechanism that controls biliverdin transportation from the shell gland into the uterus fluid, thereby playing a key role in regulating duck eggshell color.

The nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) triggers adipocyte differentiation by regulating lipogenic genes. A ligand for PPARgamma is necessary to activate PPARgamma function. Fatty acids are potential ligands for PPARgamma activation. The current experiment was designed to determine the potential for individual fatty acids to activate porcine PPARgamma ectopically expressed in myoblasts. The expression of adipocyte fatty acid binding protein (aP2) and adiponectin in myoblasts stably expressing porcine PPARgamma was increased when docosahexaenoic acid (DHA) was added to the adipogenic medium. The response was positively related to DHA concentration and suggests that DHA may bind to and activate porcine PPARgamma, leading to increased expression of aP2 and adiponectin. The conditioned media collected from myoblasts expressing PPARgamma between d 3 and 6 or between d 6 and 9, but not DHA itself, activated the aP2 gene promoter-driven luciferase activity. These results suggest that a metabolite of DHA is the ligand binding to and activating porcine PPARgamma. The metabolite and pathway for its production are currently unknown.

The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P0.05, log(2)or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

The human Enterovirus genus of the piconavirus family causes most of the febrile illnesses that affect children during the summer season in Taiwan. Enterovirus type 71 (EV71) plays a key role in patients with hand-foot-and-mouth disease (HFMD) combined with severe paralysis or encephalitis. It is important to find a method for preventing infection with EV71 since there is no antiviral agent or vaccine for humans. In this study, we developed a transgenic mouse model for demonstrating the protective effects of recombinant lactoferrin (LF) against EV71 infection. Transgenic mice carrying alpha-lactalbumin-porcine lactoferrin (alphaLA-pLF) and BALB/c wild-type mice were subjected to EV71 inoculation. First, we analyzed the expression efficiencies of recombinant pLF (rpLF) in hemizygous and homozygous transgenic mice. Following EV71 inoculation on the 4th day of life, pups ingesting transgenic milk showed the significantly higher survival rate and heavier body weight compared to wild-type mice. RT-PCR analysis for EV71 viral RNA showed that the recombinant pLF had a blocking effect on EV71 infection. Our data suggest that oral intake of pLF-enriched milk exhibited the ability to prevent infection with EV71. The study also provides an animal model for validating the protective effects of pLF.

The purpose of this study was to detect expression of genes related to egg production in Taiwan Country chickens by suppression subtractive hybridization. Liver samples of mRNA extraction from two Taiwan Country chicken strains (L2 and B), originated from the same population but with very distinct egg production rates after long-term selection for egg and meat production respectively. Two-way subtraction was performed. The hepatic cDNA from the low egg production chickens (B) was subtracted from the hepatic cDNA from the high egg production strain (L2). The reversed subtraction (L2 from B) was also performed. The resulting differentially expressed gene fragments were cloned and sequenced. We sequenced 288 clones from the forward subtraction and 96 clones from the reverse subtraction. These genes were subjected to further screening to confirm the differential expression between the two genetic breeds of chickens. The apolipoprotein B (apoB) was expressed to a greater extent in the liver of the L2 than in the B line chickens. The 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (PURH) was expressed to a greater extent in the liver of the B than in the L2 strain chickens. We demonstrated that both apoB and PURH were more highly expressed in the liver than that in other tissues (muscle, ovary, and oviduct) in laying Taiwan Country chickens. Taken together, these data suggest that after the selection for egg production, expression of apoB and PURH genes were also changed. Whether the changed expression of these genes is directly related to egg production is not known, but these two genes may be useful markers for egg laying performance in Taiwan Country chickens.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical role in regulating adipogenesis. The expression of peroxisome proliferator-activated receptor delta (PPARdelta) precedes that of PPARgamma during adipocyte differentiation in rodents. The current experiment was designed to study the function of porcine PPARdelta and the interaction of PPARdelta and PPARgamma in adipocyte differentiation. Inhibition of myogenesis was observed in mouse myoblasts expressing porcine PPARdelta, similar to myoblasts expressing PPARgamma. Treatment of myoblasts expressing PPARdelta with ligands for both PPARdelta and PPARgamma enhanced lipogenesis and adipogenesis to a greater extent than treatment with a PPARgamma ligand alone, suggesting that both genes were involved in regulating lipogenesis and adipogenesis. The ability to transdifferentiate myoblasts into adipocytes was decreased in myoblasts coexpressing PPARdelta with either wild type or mutated PPARgamma (Ser 112 was mutated to Ala; the mutated PPARgamma is more active than the wild type) compared with myoblasts expressing PPARgamma alone. Adipocyte differentiation in myoblasts coexpressing PPARdelta and mutated PPARgamma was greater than in myoblasts coexpressing PPARdelta and wild type PPARgamma, confirming that Ser 112 is important for the function of PPARgamma. Taken together, our results demonstrate that overexpression of PPARdelta inhibits myotube formation and also enhances adipocyte differentiation. However, the complexity and interaction of PPARdelta and PPARgamma in adipogenesis are not clearly understood.

We have constructed a tissue-specific in-house cDNA microarray to identify differentially expressed transcripts in shell glands from low (B) and high (L2) egg production strains of Taiwanese country chickens during their egg-laying period. The shell gland cDNA library was constructed from the high egg production strain. cDNA clones (7680) were randomly selected and their 5′-end sequences characterized. After excluding overlapping sequences, an in-house cDNA microarray, representing 2743 non-redundant transcripts, was generated for functional genomic studies. Using our microarray, we have successfully identified 85 differentially expressed transcripts from the two different strains of chicken shell glands. In this study, 34 of these transcripts were associated with signal transduction, protein biosynthesis, cell adhesion, cellular metabolism, skeletal development, cell organization and biogenesis. We selected a number of the differentially expressed transcripts for further validation using semi-quantitative RT-PCR. These included elongation factor 2 (EEF2), ovocalyxin-32 (OCX-32) and annexin A2 (ANXA2) which were expressed at high levels in the chicken shell glands of the B strain and, in contrast, the coactosin-like protein (COTL1), transcription factor SOX18 and MX protein were more highly expressed in the L2 strain. Our results suggest that these differentially expressed transcripts may be suitable to use as molecular markers for high rates of egg production, and now need to be investigated further to assess whether they can be applied for use in breeding selection programs in Taiwanese country chickens.

The European Commission has proposed a permanent ban on the use of antibiotics as an ingredient in animal feed to promote growth. Lactoferrin is a globular multifunctional protein that has been shown to play a role in iron absorption and to have antimicrobial and anti-inflammatory activities. Therefore, lactoferrin may serve as a nontherapeutic alternative to antibiotics in livestock husbandry. As a pilot study toward this goal, transgenic mice have been generated harboring a porcine lactoferrin (pLF) gene driven by the mammary gland-specific promoter of the bovine alpha-lactalbumin (alphaLA) gene. The alphaLA-pLF hybrid gene was confirmed to have been successfully integrated and transmitted stably through the germ-line in 9 (5 females and 4 males) of 14 transgenic founders. In the female progenies of six lines analyzed, the transgene copy numbers ranged from 1 to 20 with 1-4 integration sites. Significant levels of pLF protein in milk ranging from 40 to 106 microg/mL with physical characteristics similar to those of native pLF in sow's milk were achieved in three of the transgenic lines obtained. Tissue- and stage-specific pLF expressions were restricted to the mammary gland of the transgenic female mice during lactation. It was further demonstrated that the growth performance of animal pups is enhanced by directly feeding the genetically engineered milk containing enriched pLF protein in transgenic mice. Furthermore, this enhanced growth performance in suckling mice was proportional to the concentration of pLF present in milk.

Caveolin-1 of the caveolin family of proteins regulates mammary gland development and has been shown to play a contradictory role in breast tumor progression. A specific anti-Caveolin-1 antibody will be useful for functional study of Caveolin-1 in different tissues. In this study, we generated a rabbit polyclonal antibody that specifically recognizes the N-terminal amino acids 50-65 of Caveolin-1. This polyclonal antibody specifically reacted with Caveolin-1 extracted from cells of different species, including human epithelial A431 cells, goat primary mammary epithelial cells and mice fibroblast NIH 3T3 cells, by Western blotting. Endogenous Caveolin-1 protein expressing in cells and normal human tissues was detected by this polyclonal antibody using immunocytofluorescent and immunohistochemical staining, respectively. Furthermore, an apparent decrease in Caveolin-1 expression in tumorous breast and colon tissues was detected by this polyclonal antibody. In conclusion, we have identified amino acids 50-65 of Caveolin-1, which contains an epitope that is specific to Caveolin-1 and is conserved in the human, goat and mouse. In future, this anti-Caveolin-1 antibody can be used to examine the progression of breast and colon cancers and to study functions of Caveolin-1 in human, goat and mouse cells.

To study the effect of dietary docosahexaenoic acid (DHA) enrichment on the expression of hepatic genes in pigs, weaned, crossbred pigs (30 d old) were fed diets supplemented with either 2% tallow or DHA oil for 18 d. Hepatic mRNA was extracted. Suppression subtractive hybridization was used to explore the hepatic genes that were specifically regulated by dietary DHA enrichment. After subtraction, we observed 288 cDNA fragments differentially expressed in livers from pigs fed either 2% DHA oil or 2% tallow for 18 d. After differential screening, 7 genes were found to be differentially expressed. Serum amyloid A protein 2 (SAA2) was further investigated because of its role in lipid metabolism. Northern analysis indicated that hepatic SAA2 was upregulated by dietary DHA enrichment (p

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.

Adipose tissue plays a critical role in metabolism, storage, and release of fatty acids in mammals. Construction of a full-length cDNA library is an effective way to understand the functional expression of genes in adipose tissue, and in addition, novel genes for further research can be found in the library. In this study, adipose tissue RNA was extracted from three 18-mo-old Lee-Sung pigs. The mRNA was isolated, reverse transcribed, and used to construct a cDNA library. After transformation, 2,880 clones were selected and sequenced. Cluster analysis was performed, and the assembled contig of each cluster was subjected to search against DNA sequences in the nucleotide databases (NCBINR/TIGRGI). These sequences were clustered into 1,527 unique sequences; 80% of the sequences were categorized as known genes, and 20% of the sequences were categorized as unknown genes. In this adipose tissue cDNA library, approximately 16% of the genes contained full-length sequences with start and stop codons. Gene ontology analysis was performed to indicate the possible functions of these genes. Genes associated with mitochondrial function were abundant and represented 10% of the total. Several fatty acid transport genes and stearoyl coenzyme A desaturase were among the most abundant genes expressed. Tissue distribution of several abundant genes was analyzed by northern analysis, and many of these genes were transcribed in porcine adipose tissue in high copy number. Our full-length sequence data and tissue distribution data can be used to decipher the functional roles exhibited by the adipocyte under various perturbations via endocrine, environmental, genetic, nutritional, pharmacological, or physiological manipulations.

Proliferins (also termed mitogen-regulated proteins; MRP/PLFs) belong to the prolactin gene family. Mrp/Plfs are involved in angiogenesis of the uterus and placenta and maximally expressed during midgestation and decline through the remainder of the gestation period in mouse placenta. The tissue expressions of Mrp/Plfs are mainly documented in placenta, hair follicles of skin and in wound healing. In this report, we demonstrate that Plf1, Plf1 minus exon3, Plf2 and Mrp3 but not Mrp4 are expressed in mouse whole brain by diagnostic RT-PCR and Western blotting. The expression levels of Mrp/Plf mRNAs in mouse brains were low during the neonatal period, but higher in embryonic and adult stages, indicating Mrp/Plfs expression profiles are different in mouse brain and placenta. Interestingly, endogenous Mrp/Plfs were detected using immunostaining both in mouse brain sections and the neuroblastoma cell line, Neuro-2a cells. The function of PLF1 was explored by expressing exogenous PLF1 in Neuro-2a cells. This resulted in increased microvilli. Neuro-2a cells with stable expression of PLF1 had increased proliferation compared with normal and stable expressing EGFP cells when cell reached saturation density. Together these data, strongly suggest that MRP/PLFs mediate microvilli formation and contribute to cell proliferation of neuroblastoma cells.

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.

Steroid hormones and their receptors play an important role in reproductive process. Estrogen is intimately involved with pregnancy and its function is mediated through the estrogen receptor which has been chosen as a candidate gene to study litter size in pigs. In this study, we report that two estrogen receptor variants, designated pER-1 and pER-2 were co-expressed in the uteri of normal cycling Lan-Yu pig (Sus vittatus; a small-ear miniature in Taiwan) with the pER-1 expression level appeared to be several times higher than that of pER-2. These receptor variants were isolated using reverse transcription-PCR from the pig uteri and their sequences were determined. The pER-1 and pER-2 sequences, which are homologous to those found in other mammalian estrogen receptors, encode putative proteins consisting of 574 and 486 amino acids, respectively. A deletion in exon I was identified in both sequences, with deletion lengths of 63 bp in pER-1 and 327 bp in pER-2. The deletion in pER-1 is internal to that in pER-2 and both deletions resulted in a truncation of the B domain, which confers the transactivating activity of estrogen receptor protein. This result describes the existence of estrogen receptor variants with a deletion in exon I and implies the possibility that physiological functioning of an estrogen receptor may not require the presence of an intact B domain.

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine α-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The αLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 μg/ml, over 35–200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.

Recent advances in recombinant technology make transgenic animals that produce pharmaceutical proteins in their milk more feasible. The group 5 allergen isolated from Dermatophagoides pteronyssinus (Derp5) is one of the most important dust mite allergens in humans. The aims of this study were to develop transgenic mice that could secrete recombinant Derp5-containing milk and to demonstrate that ingesting recombinant milk protects against allergic airway inflammation. Two transgenes were constructed separately. The α-LA-Derp5f transgene consisted of the bovine α-lactalbumin (α-LA) promoter and full-length Derp5 cDNA. The α-LA-CN-Derp5t transgene included the α-LA promoter, a leader sequence of αS1-casein (CN), and signal peptide-truncated Derp5 cDNA. Both species of transgenic mice were confirmed to have successful transgene integration and stable germline transmission. Western blot analysis of the milk obtained from the offspring of transgenic mice demonstrated that recombinant Derp5 was secreted successfully in the milk of αLA-CN-Derp5t transgenic mice but not in that of αLA-Derp5f transgenic mice. This study provides new evidence that transgenic mice can secrete recombinant Derp5 efficiently in milk by adding a signal peptide of αS1-casein. The antigenic activity of recombinant Derp5 milk was demonstrated to have a protective effect against allergic airway inflammation in a murine model in which the ingestion of recombinant Derp5-containing milk was used as pretreatment.

Competition between humans and livestock for cereal and legume grains makes it challenging to provide economical feeds to livestock animals. Recent increases in corn and soybean prices have had a significant impact on the cost of feed for pig producers. The utilization of byproducts and alternative ingredients in pig diets has the potential to reduce feed costs. Moreover, unlike ruminants, pigs have limited ability to utilize diets with high fiber content because they lack endogenous enzymes capable of breaking down nonstarch polysaccharides into simple sugars. Here, we investigated the feasibility of a transgenic strategy in which expression of the fungal cellulase transgene was driven by the porcine pancreatic amylase promoter in pigs. A 2,488 bp 5′-flanking region of the porcine pancreatic amylase gene was cloned by the genomic walking technique, and its structural features were characterized. Using GFP as a reporter, we found that this region contained promoter activity and had the potential to control heterologous gene expression. Transgenic pigs were generated by pronuclear microinjection. Founders and offspring were identified by PCR and Southern blot analyses. Cellulase mRNA and protein showed tissue-specific expression in the pancreas of F1 generation pigs. Cellulolytic enzyme activity was also identified in the pancreas of transgenic pigs. These results demonstrated the establishment of a tissue-specific promoter of the porcine pancreatic amylase gene. Transgenic pigs expressing exogenous cellulase may represent a way to increase the intake of low-cost, fiber-rich feeds.

Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN) is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation betweenOPNmRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcineOPNwas frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that theOPNpromoter region −2615 to −2239 nucleotides (nt) may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation ofOPNpromoter was found in dose-dependent response of 5-aza-dc treatment and followed theOPNmRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation ofOPNpromoter activity. These data suggested that methylation in theOPNpromoter plays a crucial role in the regulation ofOPNexpression that we found in cloned pigs genome.

Progress in the understanding of early mammalian embryo development has been severely hampered by scarcity of study materials. To circumvent such a constraint, we have developed a strategy that involves a combination of in silico mining of new genes from expressed sequence tags (EST) databases and rapid determination of expression profiles of the dbEST-derived genes using a PCR-based assay and a panel of cDNA libraries derived from different developmental stages and somatic tissues. We demonstrate that in a random sample of 49 independent dbEST-derived zinc finger protein genes mined from a mouse embryonic 2-cell cDNA library, more than three-quarters of these genes are novel. Examination of characteristics of the human orthologues derived from these mouse genes reveals that many of them are associated with human malignancies. Expression studies have further led to the identification of three novel genes that are exclusively expressed in mouse embryos before or up to the 8-cell stage. Two of the genes, designated 2czf45 and 2czf48 (2czf for 2-cell zinc finger), are zinc finger protein genes coding for a RBCC protein with a RFP domain and a protein with three C2H2 fingers, respectively. The third gene, designated 2cpoz56, codes for a protein with a POZ domain that is often associated with zinc finger proteins. These three genes are candidate genes for regulatory or other functions in early embryogenesis. The strategy described in this report should generally be applicable to rapid and large-scale mining of other classes of rare genes involved in other biological and pathological processes. Mol. Reprod. Dev. 59:249–255, 2001. © 2001 Wiley-Liss, Inc.

Ribosomal RNA large subunit methyltransferase J (RrmJ), an Escherichia coli heat shock protein, is responsible for 2′-O-ribose methylation in 23S rRNA. In mammals, three close homologs of RrmJ have been identified and have been designated as FTSJ1, FTSJ2 and FTSJ3; however, little is known about these genes. In this study, we characterized the mammalian FTSJ2, which was the most related protein to RrmJ in a phylogenetic analysis that had similar amino acid sequence features and tertiary protein structures of RrmJ. FTSJ2 was first identified in this study as a nucleus encoded mitochondrial protein that preserves the heat shock protein character in mammals in which the mRNA expressions was increased in porcine lung tissues and A549 cells after heat shock treatment. In addition, a recent study in non-small cell lung cancer (NSCLC) suggested that the FTSJ2 gene is located in a novel oncogenic locus. However, our results demonstrate that the expression of FTSJ2 mRNA was decreased in the more invasive subline (CL1-5) of the lung adenocarcinoma cells (CL1) compared with the less invasive subline (CL1-0), and overexpression of FTSJ2 resulted in the inhibition of cell invasion and migration in the rhabdomyosarcoma cell (TE671). In conclusion, our findings indicate that mammalian FTSJ2 is a mitochondrial ortholog of E. coli RrmJ and conserves the heat shock protein properties. Moreover, FTSJ2 possesses suppressive effects on the invasion and migration of cancer cells.

A specific antiserum against the porcine sperm motility inhibitor (SMI) was used in Western blotting analysis of tissue homogenates to reveal the possible origin of SMI in the boar reproductive system at different ages. The ages of the boar used were day 0, day 15, day 30, day 60, day 100, day 120, day 135, day 150, and day 210. The tissue homogenates of the day 60 and older showed immunoreaction. The results were further checked by indirect immunohistochemical staining and observed under light microscope. The SMI antigen appeared in the epithelial cells and in the lumen of the secretory ducts of the prostate gland. These results indicate that porcine SMI is synthesized only by the postnatal prostate gland. The homogenate of the prostate gland of day 100 was also used for the purification of SMI. The prostatic SMI was co-eluted with the seminal SMI in the reversed phase HPLC. Mass spectrometric analysis of the prostatic SMI revealed a molecular weight of 10,066. These results indicate that the prostatic SMI is identical to that purified from seminal plasma (Jeng et al., 1993; Biochem Biophys Res Communi 191:435-440).

OBJECTIVE: It is not known whether progesterone, which plays a key role in establishing and maintaining pregnancy, acts directly on embryos or indirectly through the mother's reproductive tract. Since the physiological effects of progesterone are mediated by progesterone receptors (PR), the expression of PR during the preimplantation stages of pig embryos was determined. DESIGN AND METHODS: Preimplantation pig embryos at different developmental stages were examined using reverse transcription-polymerase chain reaction techniques for the purpose of determining PR gene expression. Immunocytochemistry procedures were used to determine whether PR mRNA is translated into PR protein in preimplantation embryos. RESULTS: PR mRNA was found in pig embryos at the two-cell stage, but levels started to decline at the four-cell stage; none was detected at the five- to eight-cell stage, nor at any time during the morula and blastocyst stages. Results showed that PR protein was immunostained in pig oocytes and embryos at the 4-cell stage, but that no significant immunostaining occurred during the morula and blastocyst stages. CONCLUSION: These results indicate that the effects of PR on early embryogenesis appear to be indirect, perhaps via PR-regulated growth-promoting factors produced in the maternal reproductive tract.

In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine. A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide. After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein. Over 95% of rGH was shown to be export into the culture supernatant. Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran. Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks. The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage. The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group. These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens.

As a first step toward elucidation of the action of factors secreted by the epithelium of oviduct, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used in this study to identify transcripts of such oviductal factors in gilts carrying various stages of early embryo development post hormone-induced ovulation. A total of 13 differentially expressed transcripts were identified between 50 and 120 hr post-hCG injection (between 1- and 8-cell embryonic stages). Twelve of these transcripts were found to be initially expressed at 96 hr post-hCG injection (at 4-cell embryonic stage) and beyond. Three of such genes were shown by sequence analysis to be the porcine transforming growth factor-alpha, the porcine transforming growth factor-beta-binding protein II and a porcine astral natriuretic factor receptor-like transcript. Only one differentially expressed gene was detected between 50-60 and 85 hr post-hCG injection, and this gene turned out to be the porcine follicle-stimulating hormone receptor. The remaining eight transcripts detected by DDRT-PCR were novel. Moreover, most of these newly expressed genes were found to be turned on at a time coincidental with that of the 4-cell block of porcine embryos cultured in vitro. Our results demonstrate that DDRT-PCR is a feasible approach for rapid identification of genes that are differentially expressed in oviductal epithelium. Some of the genes thus identified may be important for unhindered development of embryos in the oviduct.