Optimization of human recombinant granulocyte-colony stimulating factor supplementation during in vitro production of porcine embryos to improve the efficiency of resource utilization of poor-quality cumulus-oocyte complexes.

Granulocyte colony-stimulating factor (G-CSF), a pleiotropic cytokine, is secreted by the reproductive tract. Furthermore, our previous study indicated that human recombinant G-CSF (hrG-CSF) supplementation during porcine oocyte in vitro maturation (IVM) or during embryo in vitro culture (IVC) improved their quality and development potential when using cumulus-oocyte complexes (COCs) with more than three cumulus cell layers (CCL >3). Thus, in this study, we investigate the optimal conditions of hrG-CSF supplementation throughout the in vitro production (IVP: IVM + IVC) system to improve the embryo production efficiency of "poor-quality (CCL ≤3)" oocytes. COCs were classified into two groups according to the number of CCL (>3 and ≤3) and embryonic viability was analyzed after treatment with hrG-CSF during IVC. The mRNA transcription levels of G-CSF in COCs were compared based on their type and the period of IVM. Finally, developmental capacity and quality were evaluated after treatment with hrG-CSF for different periods of IVP. No marked effects on the developmental potential of embryos when using CCL ≤3 type COCs were observed after supplementing hrG-CSF only during IVC. Moreover, the mRNA transcription level of G-CSF increased gradually with IVM culture time and was higher in CCL ≤3 COCs than in >3. Supplementing hrG-CSF only during the IVM period resulted in the best embryo developmental potential, while supplementing hrG-CSF during the IVP period resulted in the best quality embryos, reflected in the increased total cell number and decreased apoptotic nuclei index of blastocysts. These findings indicate that "poor-quality" COCs may have a greater demand for G-CSF than "good-quality", meanwhile hrG-CSF supplementation throughout IVP improves resource utilization efficiency in poor-quality COCs.

Ancient DNA Reveals Maternal Philopatry of the Northeast Eurasian Brown Bear (Ursus arctos) Population during the Holocene.

Significant palaeoecological and paleoclimatic changes that took place during Late Pleistocene-Early Holocene transition are considered important factors that led to megafauna extinctions. Unlike many other species, the brown bear (Ursus arctos) has survived this geological time. Despite the fact that several mitochondrial DNA clades of brown bears became extinct at the end of the Pleistocene, this species is still widely distributed in Northeast Eurasia. Here, using the ancient DNA analysis of a brown bear individual that inhabited Northeast Asia in the Middle Holocene (3460 ± 40 years BP) and comparative phylogenetic analysis, we show a significant mitochondrial DNA similarity of the studied specimen with modern brown bears inhabiting Yakutia and Chukotka. In this study, we clearly demonstrate the maternal philopatry of the Northeastern Eurasian U. arctos population during the several thousand years of the Holocene.

Comparison of pregnancy rate in dromedary camel between early-stage embryos and blastocyst transfer produced by somatic cell nuclear transfer using in vitro-matured oocytes.

We compared the pregnancy and live birth rates following transfer of early-stage embryos or blastocysts produced by somatic cell nuclear transfer using in vitro-matured oocytes. In total 102 ovaries were collected from dromedary camels at a local abattoir; from these 1048 cumulus-oocytes complexes (COCs) were aspirated and cultured for 42 h in a commercial maturation medium. Metaphase II oocytes were subjected to nuclear transfer. Somatic cell nuclear transfer-derived embryos were cultured in a commercial embryo medium for 2 or 7 days. Next, 71 early-stage embryos were surgically transferred to the left fallopian tube of 28 recipients and 47 blastocysts were transferred to the left uterine horn of 26 recipients. Early pregnancy was detected by serum progesterone (P4), and pregnancy was confirmed using ultrasonography on days 30 and 90 after embryo transfer. Pregnancy rate based on P4 level was 17.86% (5/28) and 11.54% (3/26) for early-stage embryo and blastocyst transfer, respectively. In the early-stage embryo group, out of five recipients, one recipient had lost the pregnancy by the first ultrasonography on day 30; two other recipients aborted at 14 and 24 weeks, and two recipients gave live births. In the blastocyst group, out of three recipients, one lost the pregnancy at an early stage and two recipients gave live births. Therefore, for dromedary camels, we recommend transvaginal blastocyst transfer from the standpoint of the pregnancy and live birth rate, ease of the transfer procedure, and comfort and safety of the recipients.

The Resurrection of Mabrokan: Production of Multiple Cloned Offspring from Decade-Old Vitrified Tissue Collected from a Deceased Champion Show Camel.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to reproduce animals with superior genetics. Viable cell lines are usually established from tissues collected by biopsy from living animals in the SCNT program. In the present study, tissues were collected and preserved from a suddenly deceased champion camel. We established cell lines from these decade-old tissues and used them as nuclear donors. After 42 h of in vitro maturation, 68.00 ± 2.40% of oocytes reached the metaphase II (M II) stage while 87.31 ± 2.57% in vivo collected oocytes were matured at collection (p < 0.05). We observed a higher blastocyst formation rate when in vivo matured oocytes (43.45 ± 2.07%) were used compared to in vitro matured oocytes (21.52 ± 1.74%). The live birth rate was 6.45% vs. 16.67% for in vitro and in vivo matured oocytes, respectively. Microsatellite analysis of 13 camel loci revealed that all the SCNT-derived offspring were identical to each other and with their somatic cell donor. The present study succeeded in the resurrection of 11 healthy offspring from the decade-old vitrified tissues of a single somatic cell donor individual using both in vitro and in vivo matured oocytes.

Effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro culture on porcine pre-implantation embryos.

Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte and embryo developmental competence in humans. The aim of our study was to determine whether CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos. To do this, we first performed reverse-transcription polymerase chain reaction (RT-PCR). Second, we performed parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) to evaluate the embryonic developmental potential after hrG-CSF supplementation based on various concentrations (0 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL) and durations (Un-treated, Days 0-3, Days 4-7, and Days 0-7) of IVC. Finally, we examined transcriptional levels of several marker genes in blastocysts. The results of our study showed that CSF3 transcript was present in all samples we assessed. CSF3-R was also detected, except in cumulus cells and blastocysts from PA. Furthermore, 10 ng/mL and Days 0-7 were the optimal concentration and duration for the viability of in vitro embryonic development, especially for SCNT-derived embryos. The rate of blastocyst formation and the total cell number of blastocysts were significantly enhanced, while the number and index of apoptotic nuclei were significantly decreased in optimal condition groups compared to others. Moreover, the transcriptional levels of anti-apoptotis- (BCL2), proliferation- (PCNA), and pluripotency- (POU5F1) related genes were dramatically upregulated. In conclusion, for the first time, we demonstrated that CSF3 and CSF3R were expressed in porcine reproductive organs, cells, and embryos. Additionally, we determined that hrG-CSF treatment improved porcine embryonic development capacity in vitro.

Trehalose supplementation during porcine oocytes in vitro maturation improves the developmental capacity of parthenotes.

Autophagy is a critical process in early mammalian embryogenesis. Mammalian target of rapamycin (mTOR) inhibitors are major regulators of autophagy. However, mTOR plays a vital role in major signaling pathways controlling cell growth and metabolism; thus, more secure autophagy activation methods should be considered. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Trehalose treatment during in vitro maturation (IVM) did not affect the nuclear maturation rates of oocytes. Oocytes treated with 25 mM trehalose during IVM had a significantly higher (P < 0.05) blastocyst formation rate (64.2%) after PA compared to that in control oocytes (52.0%). Blastocyst quality was also improved in the trehalose-treated group. The total cell numbers for blastocyst formation and expanded blastocyst formation were significantly increased in the trehalose-treated group (52.2% and 27.7%, respectively) compared to those in the control group (36.9% and 11.0%, respectively). Trehalose treatment led to the increased expression of LC3, an autophagy marker, in metaphase II oocytes and 4-cell stage embryos. Gene expression analysis revealed that the expression of several autophagy related genes (LAMP2, pATG5, and LC3) increased, while the Bax/Bcl2 ratio and pro-apoptotic Bak transcript levels were decreased in the trehalose-treated group. In conclusion, these results indicate that treatment with trehalose during IVM improved the developmental potential of porcine embryos by down-regulation of pro-apoptotic genes and up-regulation of autophagy-related genes and marker. Trehalose may be useful for the large-scale production of high-quality porcine blastocysts in vitro.

Optimal Fixation Location in Intraoral Reduction Malarplasty Using an L-Shaped Osteotomy.

BACKGROUND: Reduction malarplasty, popular among Asians, is currently performed preferably via the intraoral approach. However, to prevent complications such as malunion and cheek drooping, a rigid fixation is essential to compensate for the action of the masseter. Accordingly, the authors aimed to perform an experimental study on the optimal fixation location during intraoral reduction malarplasty using an L-shaped osteotomy. METHODS: Fixation points that are not difficult to access during an L-shaped osteotomy were selected as 1 point in the freed zygomaticomaxillary complex and 3 points in the medial part of the malar complex (low, middle, and high levels).After setting the fixation points in the medial malar complex and lateral segment of skull replicates, resistance to traction was measured at each level, and maxillary bone thickness was also measured at each level at the fixation points of the medial malar complex in a cadaveric head. RESULTS: In the medial malar complex, resistance to traction increased at higher levels, while the maxillary bone was much thicker at the high level than at the low-middle level. CONCLUSION: This study demonstrated that it is better to locate the fixation point at a higher level in the medial part of the malar complex, closer to the lateral orbital rim.

Reconstruction of extended orbital floor fracture using an implantation method of gamma-shaped porous polyethylene.

BACKGROUND: The conventional surgical method for reconstructing orbital floor fractures involves restoration of orbital continuity by covering an onlay with a thin material under the periorbital region. However, in large orbital floor fractures, the implant after inserting is often dislocated, leading to malposition. This study aimed to propose a novel implanting method and compare it with existing methods. METHODS: Among patients who underwent surgery for large orbital floor fractures, 24 who underwent the conventional onlay implanting method were compared with 21 who underwent the novel γ implanting method that two implant sheets were stacked and bent to resemble the shape of the Greek alphabet γ. When inserting a γ-shaped implant, the posterior ledge of the orbital floor was placed between the two sheets and the bottom sheet was impacted onto the posterior wall of the maxilla to play a fixative role while the top sheet was placed above the residual orbital floor to support orbital contents. Wilcoxon signed-rank test and Mann-Whitney U test were used for data analyses. RESULTS: Compared to the conventional onlay method, the gamma method resulted in better restoration of orbital contents, better improvement of enophthalmos, and fewer revision surgeries. CONCLUSION: Achieving good surgical outcomes for extended orbital floor fractures is known to be difficult. However, better surgical outcomes could be obtained by using the novel implantation method of impacting a γ-shaped porous polyethylene posteriorly.

Two separate nodules of Merkel cell carcinoma occurring concomitantly on one cheek.

Merkel cell carcinoma (MCC) is a rare and highly aggressive neuroectodermal carcinoma arising from mechanoreceptor Merkel cells. Multiple MCCs are even rarer. We report a case of two independent MCCs simultaneously present in the cheek of a patient, which were effectively and esthetically treated using a cheek flap. Punch biopsy performed in a 60-year-old woman admitted with a chief complaint of two skin-colored hard nodules in her left cheek, accompanied by an itching sensation, was suggestive of MCC. Accordingly, we performed sentinel lymph node biopsy through the modified Blair incision under general anesthesia, in cooperation with the head and neck surgery department. The defect was covered with a cheek flap by slightly extending the existing incision following wide excision with a safety margin of 1 cm. This paper is significant in that it introduces an effective reconstruction technique that maintains function using a cheek flap for the management of this rare case. In addition, this paper is the first to classify multiple MCCs according to the time of onset. We believe that this paper presents an effective alternative reconstruction technique with sentinel node biopsy through the modified Blair incision.

Delayed Reduction of Zygomatic Arch Fracture: Effectiveness of the Rowe Zygoma Elevator.

The zygomaticomaxillary complex fractures are common fractures among the all facial fractures, and the zygomatic arch fracture accounts for approximately 10% of all facial fractures. Various routes (Gillies app, Direct cutaneous app, Bicoronal, intraoral) and devices (Foley's ballon catheter, Rowe zygoma elevator, Langenbach, deschamps needle holder, Bone hook) for a surgical intervention of the zygomatic arch have been introduced. However, no study exists that has described an effective method of surgery for zygomatic arch fractures that have been left untreated for a long period, or for unstable reduction that have not been reduced >1 month after surgery. The authors describe the case of a 53-year-old female patient who was diagnosed an unstable reduction of the zygomatic arch, 6 weeks after undergone open reduction and internal fixation for a zygomaticomaxillary complex fracture. The authors had successful outcome for delayed reduction of a zygomatic arch fracture using a Rowe zygoma elevator. And the authors suggest that the Rowe zygoma elevator is an easy and effective method for delayed reduction of a zygomatic arch fracture.

Forehead reconstruction using modified double-opposing rotation-advancement flaps for severe skin necrosis after filler injection.

Varying degrees of complications can occur after hyaluronic acid filler injections. Tissue necrosis due to interruption of the vascular supply is an early complication that can be severe. If the site of tissue necrosis due to the filler injection is the forehead, successfully reconstructing the region without distorting the key landmarks is challenging. We describe the case of a 50-year-old man who experienced widespread forehead skin necrosis after hyaluronic acid filler injection in the glabellar area. We successfully covered the forehead area with a 3×4-cm2 midline necrotic tissue using the modified double-opposing rotation-advancement flap method. Although modified double-opposing rotation-advancement flap closure has the disadvantage of leaving a longer scar compared to conventional double-opposing rotation-advancement flap closure, the additional incision line made along the superior border of the eyebrow aids in camouflaging the scar and decreases eyebrow distortion. Therefore, it is believed that the modified double-opposing rotation-advancement flap technique is an excellent tool for providing adequate soft tissue coverage and minimal free margin distortion when reconstructing widespread skin necrosis in the central mid-lower forehead that can occur after filler injection in the glabellar area.

Immortalization of Porcine 11ß-Hydroxysteroid Dehydrogenase Type 1-Transgenic Liver Cells Using SV40 Large T Antigen.

Cortisol is a steroid hormone essential to the maintenance of homeostasis that is released in response to stress and low blood glucose concentration. Cortisol is converted from cortisone by 11ßhydroxysteroid dehydrogenase type 1 (HSD11B1). It has been reported that too much cortisol or overexpression of HSD11B1 induces obesity and the insulin resistance that accompanies metabolic syndrome in rodent adipose tissue. In our previous study, HSD11B1-transgenic (TG) fibroblasts were established, and a porcine model was generated by SCNT using those fibroblasts. Hepatocytes overexpressing HSD11B1 were obtained from livers of this porcine model and cultured in vitro. However, the primary hepatocytes were found to have a short life span or low proliferation rate. To overcome these problems, the SV40 large T antigen was transduced into primary HSD11B1-TG hepatocytes, and those cells were immortalized. Immortalized HSD11B1-TG hepatocytes showed restored morphology, more rapid proliferation rate, and more expression of HSD11B1 than primary hepatocytes. As well, these cells kept the hepatic characteristics such as gluconeogenic response to cortisone and increased expression of hepatic makers. The immortalized HSD11B1-TG hepatocytes may be useful for studying traits and potential therapeutic drugs for treatment of metabolic disorders induced by overexpression of HSD11B1.

Generation of transgenic fibroblasts producing doxycycline-inducible human interferon-α or erythropoietin for a bovine mammary bioreactor.

Interferon α (IFN-α) is a cytokine, produced predominantly in immune cells in response to pathogens, which interferes with viral replication in host cells. Another cytokine hormone, erythropoietin (EPO), is synthesized in interstitial fibroblasts of the kidney and acts as a stimulator for the production of red blood cells. Importantly, the two cytokines have been used in the treatment of certain hematological malignancies, including renal anemia. In the production of recombinant proteins, a transgenic expression system in bovine species is an efficient strategy for pharmaceutical production. In the present study, recombinant constructs capable of producing recombinant human IFN-α and EPO proteins were established and were generated containing the mammary gland-specific αS1-casein promoter region (between -175 and + 796 nt), as this promoter was revealed to have the highest level of activity in a previous promoter study. In order to minimize developmental toxicity by constitutive exogenous expression, a doxycycline (dox)-inducible system was introduced to the IFN-α/EPO-expressing constructs. Therefore, a unitary tetracycline (tet)-on the IFN-α/EPO vector was established, which combined a tet-on activator cassette controlled by the αS1-casein promoter, with a responder cassette encoding the IFN-α/EPO gene, controlled by the tetracycline response element (TRE) promoter. In these systems, the tet-controlled transactivator is affected by mammary gland-specific αS1-casein promoter, and binding of the transcriptional activator to the TRE results in transcription of the downstream IFN-α/EPO genes in the presence of dox. To assess this, the unitary tet-on IFN-α/EPO vector was introduced into a bovine mammary gland cell line (MAC-T), and the cells were then treated with 0.1-1 µg/ml dox. A marked increase was observed in the expression levels of IFN-α/EPO. In addition, bovine transgenic fibroblasts containing a mammary gland-specific and dox-inducible IFN-α/EPO construct were generated. These transgenic fibroblasts may provide a source for somatic cell nuclear transfer for the generation of transgenic cattle producing recombinant human IFN-α/EPO protein during lactation.

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.

Generation of transgenic fibroblasts expressing pancreas­specific and doxycycline-inducible ICER Iγ for the establishment of a porcine model of human diabetes mellitus.

Inducible cyclic AMP (cAMP) early repressor (ICER) Iγ acts as an endogenous inhibitor and disrupts the transcriptional regulation of cAMP response element binding protein (CREBP) responsive genes. Since the overexpression of ICER Iγ induces severe diabetes in a transgenic mouse model, with characteristics similar to human diabetes mellitus, an ICER Iγ construct containing an adjustable pancreas tissue specific promoter was utilized in the present study. Using the human insulin promoter region, a doxycycline (dox)­inducible ICER Iγ expression system was established using the tetracycline (tet)-controlled transactivator (tTA) with a TA response element (TRE) promoter. A unitary tet-on system that combined a tet-on activator cassette was also developed and was controlled by the human insulin promoter with a responder cassette containing genes encoding ICER Iγ regulated by the TRE promoter. To determine whether dox-enhanced ICER Iγ expression affected insulin production, the unitary tet-on ICER Iγ vector was introduced into a mouse pancreatic ß-cell line and then the cells were treated with 0.1-1 mg/ml dox. The results revealed a robust increase in ICER Iγ expression and decreased insulin production. Therefore, this in vitro system may be useful for studying human diabetes mellitus and pre-diabetes using tissue-specific promoters and a dox-inducible transgene. In addition, porcine transgenic fibroblasts containing dox-inducible ICER Iγ were generated. These fibroblasts may serve as a cell source for somatic cell nuclear transfer to generate a porcine model of human diabetes mellitus.

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.

Establishment of transgenic fibroblasts for producing recombinant human interferon-α and erythropoietin in bovine milk.

Human interferon α (IFN-α) and erythropoietin (EPO) have been used for a variety of purposes in clinical medicine. Human IFN-α has been used to treat several types of viral infection and cancer, as well as renal anemia, via stimulation of erythrocyte formation in the bone marrow. Transgenic cattle are excellent candidates for pharmaceutical production for humans due to their ability to produce recombinant proteins in milk. The purpose of the present study was to generate bovine transgenic fibroblasts capable of producing recombinant human IFN-α and EPO proteins in transgenic cattle milk. First, we analyzed the promoter activities of various bovine milk protein genes in HC11 mouse mammary epithelial cells. The bovine milk protein gene promoters were cloned into the Luc gene in a promoter-less pGL3-Basic vector. Presence of the αS1-casein promoter (-175 to +796 nt) resulted in an up to 16-fold increase in luciferase activity compared with that of the promoter-less construct. In addition, the human IFN-α and EPO genes were identified as significantly overexpressed in HC11 cells compared with the promoter-less construct. Together, the present results demonstrate that the construct with the αS1-casein promoter may induce secretion of recombinant human IFN-α and EPO into bovine milk. Furthermore, we generated transgenic fibroblasts expressing human IFN-α and EPO cDNA controlled by the αS1-casein promoter and two screening markers, enhanced green fluorescent protein and neomycin resistance. These transgenic fibroblasts may be a source of somatic cells for generating transgenic cattle that produce recombinant human IFN-α and EPO proteins during lactation.

Generation of fibroblasts overexpressing liver-specific PEPCK in a miniature pig model of human type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is one of the most common complex metabolic disorders in humans, and is characterized by hyperglycemia and metabolic alterations. In T2DM, fasting hyperglycemia is attributed to excessive hepatic glucose production, and increased gluconeogenesis has been ascribed to increased transcriptional expression of phosphoenolpyruvate carboxykinase (PEPCK). In this study, we analyzed porcine PEPCK promoter activities to generate liver-specific expression vectors. We generated miniature pig fibroblasts overexpressing PEPCK via transgenes to provide an animal model of human T2DM. Various regions of the promoter showed high levels of activity in the presence of glucocorticoids, a PEPCK gene inducer, in liver cells compared to a positive control promoter. The selected promoter region for a liver-specific expression system was adopted based on the current targeting vector containing two selection markers, green fluorescence protein and a neomycin-resistance gene. The linearized vector was introduced into pig fibroblasts which facilitated liver-specific PEPCK overexpression and screening according to the two selection markers. In the present study, we used a liposome-mediated transfection protocol rather than a virus-mediated gene delivery system, since the virus may cause side effects. Following transfection, 46 colonies out of 33 transfection trials had positively integrated the overexpression vector, indicating that a relatively high transfection efficiency rate was obtained by the liposomal-mediated system. Thus, we recommend the optimal liver-specific expression system for safe and effective transfection of pig cells. We plan to use these cells for somatic cell nuclear transfer to produce piglets that overexpress liver-specific PEPCK as an animal model for human T2DM.

Establishment of inducible cAMP early repressor transgenic fibroblasts in a porcine model of human type 1 diabetes mellitus.

Diabetes mellitus is a metabolic disease caused by impaired insulin secretion from the pancreatic ß cells and increased insulin resistance in peripheral tissues. Recently, the overexpression of inducible cyclic AMP (cAMP) early repressor (ICER) Iγ in rodent pancreatic ß cells was found to induce insulin deficiency and glucagon overproduction similar to that found in human diabetes mellitus. ICER Iγ with only a DNA binding domain interrupts the transcriptional regulation of the cAMP responsive element-binding protein (CREB) target genes. Based on this information, we hypothesized that the overexpression of ICER Iγ, the most powerful competitor to CREB, could be useful for generating a pig model of diabetes. First, we evaluated the promoter activities of the human insulin gene for the ß cell-specific overexpression of ICER Iγ in the pig pancreas. The maximum promoter activity region [-1,431 nucleotides (nt) to +1 nt, +1 = the transcriptional start site] of the insulin gene presented an activity level 3-fold higher than a promoterless construct. Second, ICER Iγ overexpression controlled by this promoter region significantly blocked the glucose-mediated insulin transcription, such as that regulated by the viral promoter in the pancreatic ß cell line, MIN6. This suggests that the human insulin promoter may facilitate the overexpression of ICER Iγ in porcine pancreatic ß cells. In addition, the overexpression of ICER Iγ in porcine ß cells may induce human-like type 1 diabetes mellitus in pigs. In the present study, we generated transgenic fibroblasts containing ICER Iγ cDNA controlled by the human insulin promoter, as well as two screening markers, the green fluorescence protein and the neomycin resistance gene. These fibroblasts may provide a source for somatic cell nuclear transfer to generate a pig model that mimics human diabetes mellitus.