bluuebunny/crossref_metadata_2025 huggingface dataset:
200 SYNCHRONIZATION OF OOCYTE MEIOTIC MATURATION IN SUPEROVULATED MICE IMPROVES IN VITRO FERTILIZATION RATE
Authors: Taiyeb Ridha A. M. , Kraemer D. C.
Published: December 2011

In vitro synchronization of oocyte nuclear and cytoplasmic maturation has been found to improve the IVF rate of ovarian oocytes in several species, including humans, in comparison with nonsynchronized in vitro-matured oocytes. Here, we tested the hypothesis that synchronization of oocyte meiotic maturation by an in vivo system in superovulated mice would increase the oocyte fertilization rate when compared to that of conventional superovulated oocytes. Recently, we observed that cilostazol (CZL), a PDE3-I, was able to inhibit mouse oocyte meiotic maturation in both in vitro and in vivo systems. Administering CZL at 7.5 mg, 4 or 7 h pre-hCG allowed retrieval of ovulated oocytes of which >95% were at MI stage, scored by Nikon stereo microscope (SMZ 1500). A conventional superovulation program was adapted in all treated and their control groups, in which mice were injected with eCG and after 48 h with hCG (7.5 IU for each hormone). On the second morning, 13 to 14 h post-hCG, mice were killed and oocytes were collected from oviducts and in vitro fertilized (control). For the treated groups, CZL was administered in a single 7.5 mg oral dose (gavage) 4 or 7 h before the hCG injection. On the second morning, CZL-treated animals were killed at the same timing as control animals and oocytes were retrieved from the oviduct and in vitro matured for 6 h (for those gavaged with CZL, 4 h pre-hCG) or 3 h (for those gavaged with CZL, 7 h pre-hCG) to MII oocytes before IVF. These groups were designated as in vivo-in vitro synchronized/matured oocytes. In other groups treated with CZL, 4 or 7 h pre-hCG, the ovulated oocytes were allowed to mature in the oviduct (full in vivo synchronization and maturation) and oocytes were retrieved and fertilized with the same fertilization timings as the in vivo-in vitro synchronized/matured oocytes. Oocytes were cultured for 1 day after IVF and examined for cleavage. Statistical differences were analyzed by cross-tabulated chi-square test. The full in vivo synchronization and maturation (for both CZL dose timings of 4 and 7 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [89% (n = 219) and 92.2% (n = 374) vs 81.8% (n = 198); P = 0.034 and P < 0.0001, respectively]. The in vivo-in vitro synchronized/matured oocytes (CZL dose timing at 7 h, but not 4 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [88.5% (n = 339) vs 83.4% (n = 458), respectively; P = 0.043]. However, the increase of the IVF rate of the oocytes from mice treated with CZL, 4 h pre-hCG, in the in vivo-in vitro synchronized/matured group was not significantly different from the control group [88.5% (n = 399) vs 83.4% (n = 458), respectively; P = 0.43]. It is concluded from the present study that synchronization of oocyte meiotic maturation by the in vivo and in vivo-in-vitro protocols can increase the IVF rate of oocytes in superovulated mice.
201 EFFECTS OF REMOVING SEMINAL PLASMA DURING 8-h STORAGE BEFORE SEX-SORTING BOVINE SPERM
Authors: Burroughs C. A. , Evans K. M. , Lenz R. W. , Seidel G. E.
Published: December 2011

We evaluated sex-sorting parameters and post-thaw motility for sperm stored with or without seminal plasma for 8 h before sorting. One first ejaculate was collected from each of 6 bulls routinely collected via artificial vagina; ejaculates contained at least 70% motile and 75% morphologically normal sperm and sperm concentrations ranged from 0.75 to 2.21 × 109 sperm mL–1. Ejaculates were divided into 2 samples and centrifuged at 1000 × g for 15 min. Seminal plasma from 1 sample was replaced with TALP (pH 7.4) to a sperm concentration of 1.4 × 109 sperm mL–1. The seminal plasma/sperm admixture of the other sample (control) was suspended to initial ejaculate sperm concentration. Both samples were stored for 8 h at 16°C before being subjected to standard sex-sorting procedures. Sperm were analyzed and bulk sorted on a MoFlo SX (XY Inc., Navasota, TX, USA) flow cytometer/cell sorter for percentage of live-oriented cells, percentage of membrane-impaired sperm (cell membranes permeable to red food colouring, which were discarded during sorting) and resolution between X- and Y-bearing sperm populations (peak to valley ratio). Sorted sperm were frozen according to standard procedures and post-thaw motility was determined immediately after thawing using computer-assisted sperm analysis. Treatments were compared using a paired t-test. Control sperm stored with seminal plasma resulted in a higher percentage of live-oriented cells (55%) versus those stored without seminal plasma (51%; P = 0.02). The percentage of membrane-impaired sperm was lower for control sperm (19%) than that of samples without seminal plasma (28%; P < 0.001). Resolution was greater for sperm stored without seminal plasma (34%) than for control sperm (10%; P = 0.04). Post-thaw, both total and progressively motile sperm were higher for samples without seminal plasma (63 and 53%, respectively) compared with those of the control samples (52 and 45%, respectively; P < 0.04). In conclusion, sperm stored for 8 h without seminal plasma had greater resolution between X- and Y-bearing populations and higher post-thaw motility than control sperm. However, these samples had a higher percentage of membrane-impaired sperm that were removed during sorting. Long-term storage of sperm in their seminal plasma before sex-sorting appears to be detrimental to post-sorting, post-thaw sperm motility.
215 COMPARATIVE ANALYSIS OF PORCINE PLURIPOTENT CELL LINES DERIVED FROM SOMATIC CELLS AND VARIOUS EMBRYONIC ORIGINS
Authors: Park J. K. , Kim H. S. , Uh K. J. , Choi K. H. , Kim H. M. , Lee D. K. , Oh J. Y. , Lee C. K.
Published: December 2011

Since pluripotent cells were first derived from the inner cell mass (ICM) of mouse blastocysts, tremendous efforts have been made to establish embryonic stem cell (ESC) lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to derive pluripotent cells of naïve state that represents full pluripotency, due to the frequent occurrence of spontaneous differentiation into an EpiSC-like state during culture in pigs. We have been able to derive EpiSC-like porcine embryonic stem cell (pESC) lines of a differentiated non-ES cell state from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. In this study, we analysed characteristics such as marker expression, pluripotency and the X chromosome inactivation (XCI) status of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential; female XCI activity and a normal karyotype. Here we provide preliminary results that suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.
This work was supported by the BioGreen 21 Program (#20070401034031, PJ0081382011), Rural Development Administration, Republic of Korea.
221 GENERATION OF GERM-LINE COMPETENT EMBRYONIC STEM CELLS FROM NON-OBESE DIABETIC (NOD) MICE USING A SINGLE INHIBITOR
Authors: Liu J. , Ashton M. P. , Sumer H. , Brodnicki T. C. , O'Bryan M. K. , Verma P. J.
Published: December 2011

The use of inbred mouse strains, where selective mating and tissue availability can be used to study the role of specific genes during disease pathogenesis is a synergistic approach to human type 1 diabetes (T1D) studies, which are hindered by genetic heterogeneity and inability to obtain relevant tissue biopsies. An excellent model for T1D is the non-obese diabetic (NOD) mouse strain, which spontaneously develops autoimmunity with lymphocytic infiltration of pancreatic islets. Autoimmune disease in NOD mice is multigenic and more than 20 susceptibility loci have been identified. Thus, genetic manipulation of NOD mice via gene targeting in embryonic stem cells (ESC) is a key strategy for identifying the underlying genes for these loci and characterising their roles in disease pathogenesis. Derivation of ESCs from NOD mice has proved to be extremely challenging and the NOD mouse is classified as one of the non-permissive mouse strains for derivation of ESC (Nagafuchi et al. 1999 FEBS Lett. 455, 101–141; Nichols et al. 2009 Nat. Med. 15, 814–818). Using the conventional mouse ESC culture medium - Knockout DMED medium supplemented with 20% Knockout serum replacement (KSR), 2 mM glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol (all from Invitrogen) and 2000 U mL–1 LIF (Millipore), we efficiently derived robust NOD ESC lines by inhibiting either the ERK or the GSK3b signalling pathway. Nineteen NOD ESC lines following supplementation of the ERK signalling inhibitor SC1 (SC1-NOD ESC) and 10 lines with GSK3b signalling inhibitor BIO (designed as BIO-NOD ESC) from 22 and 12 embryos, respectively (86.3 and 83.3%; P = 0.8). Two SC1-NOD and 2 BIO-NOD ESC were selected for characterisation. All 4 lines expressed the pluripotency-specific genes OCT4, NANOG and SSEA1, showed euploid karyotypes and generated teratomas in SCID mice. Two BIO-NOD ESC lines contributed to chimeric mice after injection into C57BL/6 blastocysts and embryo transfer. Chimeras from the both BIO-NOD ESC lines produced progeny of pure NOD genetics after mating with wild-type NOD females, demonstrating germ-line competence. The NOD ESC lines generated provide a unique tool to study genes implicated in T1D.
23 DEVELOPMENTAL POTENTIAL OF CLONED TRANSGENIC PORCINE EMBRYOS PRODUCED BY SERIAL NUCLEAR TRANSFER CAN BE IMPROVED BY TREATMENT WITH HISTONE DEACETYLASE INHIBITORS
Authors: Kurome M. , Zakhartchenko V. , Kessler B. , Güngör T. , Richter A. , Klymiuk N. , Nagashima H. , Wolf E.
Published: December 2011

Recently, we generated cloned transgenic pigs by nuclear transfer (NT) using fetal fibroblasts transfected with a LEA29Y gene specifically expressed in pancreatic β-cells (INS-LEA). Transfer of 216 NT embryos into 3 recipients resulted in the birth of 9 piglets. Furthermore, we examined serial NT with donor cells of the INS-LEA cloned pigs as a means of propagating the genotype of these valuable animals. Surprisingly, no piglets were obtained after transfer of 512 NT embryos into 5 recipients, which might be due to epigenetic alterations that presumably occurred during post-implantation development of the first round cloned embryos or during nuclear reprogramming in the second round of NT. In this study we tested whether in vitro development of re-cloned embryos can be improved by their treatment with histone deacetylase inhibitors (HDACi), scriptaid and suberoylanilide hydroxamic acid (SAHA). As nuclear donors, ear fibroblast cells derived from the INS-LEA cloned pig were used. Nuclear transfer was performed using in vitro-matured oocytes as previously reported (Kurome et al. 2006, Transgenic Res. 15, 229–240). After activation, reconstructed embryos were treated immediately by scriptaid (500 nM) and SAHA (10 μM) for 16 and 10 h, respectively. Development of NT embryos was assessed by cleavage and blastocyst formation during culture for 7 days. The cell number of blastocysts was also counted after fixation and staining. There was no significant difference in the cleavage rate between treated and non-treated by both HDACi, whereas treatment of NT embryos with scriptaid or SAHA significantly enhanced their development to blastocyst compared with non-treated NT embryos (22.2%, 43/194 and 22.7%, 34/150 vs 7.7%, 15/195 and 12.3%, 18/146, respectively; P < 0.05). Notably, blastocyst rates obtained after treatment of re-cloned embryos with HDACi were similar to those in the first round of NT (21.2%, 33/156). Treatment of NT embryos with HDACi did not increase mean cell number of blastocysts compared with non-treated embryos. The results of our study show that in vitro developmental competence of embryos produced by serial NT can be improved by both HDACi used, scriptaid as well as SAHA, which has not been reported before in pig cloning. To determine the post-implantation developmental potential, re-cloned embryos treated with HDACi will be transferred to surrogate gilts.
This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung and Mukoviszidose e.V.
234 EFFICIENT EXPRESSION OF HUMAN ENDOTHELIAL PROTEIN C RECEPTOR AND HUMAN THROMBOMODULIN IN TRANSFECTED PIG PRIMARY hCD55+-GAL–/– FIBROBLASTS USING F2A EXPRESSION VECTOR
Authors: Perota A. , Lagutina I. , Colleoni S. , Duchi R. , Lazzari G. , Cozzi E. , Lucchini F. , Galli C.
Published: December 2011

The genetic engineering of the pig genome for xenotransplantation studies requires the insertion of different transgenes to create multi-transgenic pigs. In order to simultaneously add more transgene in a single genetic insertion, we constructed a polycistronic vector using the F2A self-cleaving peptide. Moreover, this solution has the added advantages of preventing possible segregation during breeding of the animals and of guaranteeing an equimolar production of chosen transgenes. The scope of this work was the construction and validation of an ubiquitous F2A-bicistronic expression vector for human thrombomodulin (hTM) and human endothelial protein C receptor (hEPCR) genes in pig primary hCD55-GAL–/– cells to establish transgenic fibroblasts colonies, to be used for somatic cell nuclear transfer (SCNT) to generate pigs for xenotransplantation research. The expression vector consisted of pCAGGS promoter (CMV-IE+chicken β actin) followed by hEPCR-furinF2A-hTM coding sequence. The resulting expression cassette was inserted between 2 insulators obtained from the 5′ MAR region of chicken lysozyme. Outside of this insulated structure, there is a loxable puromycin selection cassette. The resulting purified and linearized expression vector (pEFTM/Lgu I = 5 μg) was transfected into hCD55-GAL–/– primary fibroblasts (1 × 106), using Nucleofector (Amaxa, Lonza, Cologne, Germany), in parallel for comparative purposes we cotransfected the 2 pCAGGS-monocistronic vectors for the same transgenes (hEPCR and hTM = 1:3, 5 μg). Transfected cells were selected with puromycin (1 μg mL–1) for 15 days. After 8 days of selection, resistant colonies were picked up and expanded into 24-well plates for cryopreservation and analyses. Bicistronic transfection produced 20 clones and cotransfection only 8 clones that were analysed by Western blot (WB) and by immunocytochemistry (ICC) using polyclonal antibody anti-EPCR (1:250, R&D) and monoclonal antibody ab6980-Abcam (1:5000, Abcam, Cambridge, UK) in WB; polyclonal antibody RCR252 (1:100, Sigma-Aldrich, St. Louis, MO, USA) and monoclonal antibody ab6980-Abcam (1:100, Abcam) for ICC. Seventeen bicistronic clones (85%) and 2 cotransfected monocistronic clones (25%) were positive for both transgenes using WB. After ICC analyses, only 11 bicistronic colonies (55%) and 1 cotransfected colony (12.5%) uniformly expressed the desired transgenes and were selected for SCNT. The pCAGGS promoter maintained its strong expression also using the hEPCR-FurinF2A-hTM coding sequence and this bicistronic solution permitted us to improve our results obtained with co-transfection. Availability of hEPCR+ hTM+ hCD55+-GAL–/– colonies will allow us to obtain a new transgenic background for future xenotransplantation projects.
This study was supported by EU grant no. LSHB-CT-2006-037377 (Xenome) and by Regione Lombardia (Superpig project).
236 GENE TARGETING USING ZINC FINGER NUCLEASES (ZFN) IN THE PORCINE FIBROBLAST
Authors: Kim S. , Kim J. W. , Lee S. M. , Kim J. H. , Kang M. J.
Published: December 2011

Gene targeting is a genetic technique that utilises homologous recombination between an engineered exogenous DNA fragment and the endogenous genome of an animal. In domestic animals, gene targeting has provided an important tool for producing knockout pigs for the α1,3-galactosyltransferase gene (GGTA1) to use in xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse embryonic stem cells. The application of gene targeting to somatic cells has been limited by its low efficiency. Recently, knockout rat and mouse were generated by introducing nonhomologus end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knockout and knock-in techniques in domestic animals is very important in biomedical research. In this study, we investigated homologous recombination events at the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. The CMAH-targeted ZFN plasmid and mRNA were purchased from Sigma-Aldrich (St Louis, MO, USA). Porcine ear fibroblasts cells were obtained from a 10-day-old male Chicago miniature pigs. The fibroblasts were cultured in DMEM containing 15% fetal bovine serum, 1 × nonessential amino acids, 1 × sodium pyruvate, 10–4 M β-mercaptoethanol, 100 unit mL–1 penicillin and 100 μg mL–1 streptomycin. The cells were trypsinized and resuspended at a concentration of 1.25 × 107 cells mL–1 in F10 nutrient mixture. Four hundred microliters of the cell suspension was electroporated in a 4-mm cuvette with 4 pulses of 1 ms duration using 400V capacitive discharges using the CMAH neo targeting vector and ZFN plasmid or RNA. The CMAH neo targeting vector consists of the neomycin resistance gene (neo) as a positive selectable marker gene, 789-bp 5′ arm and 763-bp 3′ arm from exon 8 of CMAH gene. After selection of G-418, PCR analysis was performed using 64 colonies transfected with ZFN plasmid and 48 colonies transfected with ZFN RNA. As a result, 19 positive colonies were identified in colonies transfected with ZFN plasmid and 15 colonies were identified in colonies transfected with ZFN RNA. The targeting efficiency was 29.7 and 31.6% in the colonies transfected with ZFN plasmid and ZFN RNA, respectively. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in production of CMAH knockouts for xenotransplantation.
24 TARGETED DISRUPTION OF ATAXIA-TELANGIECTASIA MUTATED GENE IN MINIATURE PIGS BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Kim Y. J. , Ahn K. S. , Kim M. J. , Ahn J. S. , Ryu J. H. , Kim M. J. , Shim H.
Published: December 2011

Ataxia-telangiectasia (A-T) is a genetic disease caused by germline biallelic mutation in the ataxia-telangiectasia mutated gene (ATM) that results in partial or complete loss of ATM expression or activity. A diverse phenotype of the disease includes progressive cerebellar ataxia, oculocutaneous telangiectasias, radiation hypersensitivity, increased cancer incidence, immunodeficiency and chromosomal instability. The frequency of A-T in the United States and United Kingdom has been estimated to be 1:40 000. Heterozygous carriers may also have predisposition to diverse cancers. Although ATM-deficient mice have been produced, none reflects the extent of neurological abnormalities involving the loss of Purkinje cells of the cerebellum seen in patients. Hence, miniature pigs, which are anatomically and physiologically closer to humans, might serve as an alternative model for human A-T. In the present study, we attempted a targeted disruption of ATM in miniature pigs by somatic cell nuclear transfer. Most A-T patients possess mutated ATM with truncation, resulting in prematurely terminated ATM proteins that are highly unstable. To induce the truncation of ATM, we disrupted exon 59 region of ATM that has been known to be critical for ATM kinase activity. Miniature pig fetal fibroblasts were transfected with ATM-targeting vector and treated by neomycin for 2 weeks. A total of 139 colonies were screened by PCR and 3 among them were identified as homologous recombinants with monoallelic disruption of ATM (targeting efficiency = 2.1%). One of the 3 colonies was chosen and used for subsequent nuclear transfer. In total, 611 nuclear transfer embryos reconstructed with ATM-targeted fetal fibroblasts were transferred into 5 surrogate gilts. Two gilts became pregnant and developed to term with a total of 5 live piglets delivered. Being analysed by PCR, all piglets born were found to be ATM gene-targeted. Because conventional murine models of human A-T have provided only limited insights into therapies and pharmacological treatments, the generation of miniature pigs with disrupted ATM will allow new opportunities to more precisely understand A-T and to accelerate discovery of strategies for prevention and treatment of the disease. In addition, such animals may be utilised in studying cancer with respect to the involvement of ATM in cell cycle arrest, DNA repair and apoptosis.
THE PIG AS A BIOMEDICAL MODEL: HOW THE NATIONAL SWINE RESOURCE AND RESEARCH CENTER CAN HELP YOU
Authors: Walters Eric M. , Samuel Melissa S. , Wells Kevin D. , Riley Lela K. , Prather Randall S.
Published: December 2011

Swine are an excellent large animal model for human health and disease because their size and physiology are similar to humans, and the progression of many diseases, such as cardiovascular disease, is similar to humans. Due to these similarities, technology, procedures, and novel treatments that have been developed for human diseases can be applied to swine, and vice versa. In addition there are some human diseases, such as cystic fibrosis, that don't recapitulate in the rodent models but are well modeled in swine. Swine have been accepted as the model of choice for several medical applications such as xenotransplantation and surgical training for medical students. Because of the usefulness of swine to better understand human health, the National Institutes of Health has established the National Swine Research and Resource Center to help meet the needs of the biomedical community. While wild type swine and spontaneous mutants have been shown to be valuable, genetically engineered swine provide a new level of utility. With the improvement of the techniques to add genes and make targeted genetic modifications not only have the number of types of genetically engineered pigs increased, but also the number of different diseases modeled have increased. The completion of the swine genome combined with development and improvements of techniques for gene modification will only enhance the continued use and development of swine as models of human health, syndromes and conditions. The resources at the National Swine Resource and Research Center are available to aid investigators with their swine needs for biomedical research. Here will be discussed the importance of swine as a biomedical model, current technologies to produce genetically engineered swine, current biomedical models, and how the completion of the swine genome will promote swine as a biomedical model.
PROGRESS TOWARDS THE DERIVATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS
Authors: Petkov Stoyan , Ivics Zoltan , Niemann Heiner
Published: December 2011

Porcine induced pluripotent cells (iPSC) are considered an important large animal model for developing personalized stem cell therapies. Since the derivation of the first mouse and human iPSC, there have been relatively few reports regarding the reprogramming of pig somatic cells into pluripotency, exclusively with the use of transcription factors from human and mouse origin. To investigate whether using species-specific transcription factors would allow for an efficient reprogramming of porcine somatic cells, we have developed a Sleeping Beauty (SB) transposon system based on the porcine OCT4, SOX2, C-MYC, and KLF4 sequences and have tested it in the reprogramming of mouse and porcine fetal fibroblasts and pig EGC-like cells. Transfection of mouse embryonic fibroblasts with a multicistronic SB-tetO-pOCT4-F2A-pSOX2-T2A-pC-MYC-E2A-pKLF4-ires-Tomato vector resulted in the formation of iPSC colonies, which inactivated the exogenous transcription factors and upregulated their endogenous pluripotency genes. These cells maintained mESC-like morphology, formed embryoid bodies, and differentiated into different cell types in culture, including rhythmically contracting cardiac myocytes. In contrast, porcine fibroblasts and EGC-like cells transfected with the same transposon vector did not proliferate in culture and did not form any iPSC colonies. We then transfected these cells with multiple bi-cistronic vectors SB-Ef1a-pOCT4-ires-Tomato, SB-Ef1a-pSOX2-ires-Neo, SB-Ef1a-pC-MYC-ires-Puro, and SB-Ef1a-pKLF4-ires-Puro. As a result, both cell types formed multiple colonies with mouse ESC-like morphology. Clones established from individually picked colonies from transfected fetal fibroblasts maintained this morphology for 5-6 passages, after which they became flat and epithelial-like. They expressed endogenous SOX2, C-MYC, KLF4, and E-Cadherin, but not OCT4. At the same time, clones derived from EGC-like cells proliferated at accelerated rate and maintained their morphology for over 10 passages at the time of this writing. While the exogenous genes were expressed continuously during this period, the cells expressed also endogenous OCT4, SOX2, TERT, STELLA, TDH, and CHD1; however, C-MYC, KLF4, NANOG, and E-Cadherin expression was relatively low. These cells are currently being characterized for pluripotency. Despite the use of porcine transcription factors, the overall reprogramming of porcine cells appears to be still less efficient compared with mouse fibroblasts. Our results suggest that the species and tissue origin of the somatic cells may play a more important role in the reprogramming to pluripotency than the origin of the transcription factors used. In this respect, optimization of culture conditions may be necessary in order to allow for efficient and complete reprogramming of porcine somatic cells.
This work is supported by a research grant by the Deutsche Forschungsgemeinschaft.
25 THE DEVELOPMENTAL COMPETENCE AND PLURIPOTENT GENE EXPRESSION OF PORCINE SOMATIC CELL NUCLEUS TRANSFER EMBRYOS IMPROVED BY m-CARBOXYCINNAMIC ACID BISHYDROXAMIDE
Authors: Park S.-H. , Kim S.-Y. , Lee M.-R. , Eun H.-J. , Baik S.-K. , Kim T.-S. , Lee J.-H.
Published: December 2011

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic state after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Miyamoto et al. 2010 JBC), use of m-carboxycinnamic acid bishydroxamide (CBHA), a histone deacetylase inhibitor, during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA on the in vitro developmental competence and pluripotent gene expression of porcine SCNT embryos. The porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h) and then were assessed to cleavage rates and development to the blastocyst stage. In addition, pluripotent gene expression of SCNT embryos was analysed by RT-PCR. All data were analysed by chi-square. Following 4 replicates (207 and 124 for NT and CBHA-treated NT embryos, respectively), no significant difference was observed between NT and CBHA-treated NT embryos for cleavage rate (83.9 vs 82.2%). However, the developmental competence to the blastocyst stage differed significantly between NT and CBHA-treated NT embryos (6.8 vs 18.6%; P < 0.05). In addition, pluripotent transcription factors including Oct4, Nanog and Sox2 were higher expressed in the cloned embryos treated with CBHA (P < 0.05). The results of the present study suggest that treatment with CBHA as a histone deacetylase inhibitor significantly increased the developmental competence as well as the pluripotent gene expression of porcine SCNT embryos.
This work was partly supported by a grant from the NRF (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.
NEURODEGENERATIVE DISEASES
Authors: Chan Anthony
Published: December 2011

In the past few decades, a tremendous amount of effort has been invested in developing gene and cell therapies for inherited genetic diseases such as Huntington's disease (HD). However, progress in their clinical application has been very limited. One of the major barriers is the lack of appropriate animal models that allow precise prediction patterns in human patients. Most of the animal models used for gene and cell therapy study are primarily focused on safety and toxicity evaluation, while therapeutic efficacy cannot be fully addressed because they do not carry the same human diseases. Although mouse models of human diseases are available and have been widely used for the development of new therapies, mice are not good predictors for humans because of the fundamental differences (genome composition, body size, life span and metabolic mechanism) between humans and rodents. Although monkeys are one of the best models for studying pharmacokinetics and overall impact of treatment, they are primarily used for safety and toxicity evaluation. Even HD monkey models, created by chemical induction or focal gene transfer in the brain, develop similar cellular pathology, therapeutic efficacy and systemic evaluation cannot be determined, which is one of the major barriers in drug and therapeutic development. The development of transgenic HD monkeys has opened the door for a new paradigm of animal modeling for the advancement of novel gene and cell therapy. HD monkeys not only carry the genetic defect that leads to human HD, they also develop clinical features comparable to humans that no other animal model does. While testing in HD monkeys has yet to be achieved until a cohort of well characterized HD monkeys was established, iPS cell lines derived from HD monkeys with a board spectrum of HD pathology and clinical features are a unique in vitro model for studying HD pathogenesis and the development of novel therapeutic approaches. New knowledge and treatments generated from iPS cells can next be translated and applied in HD monkeys from whom the stem cells were derived, thus the goal of personalized medicine can also be evaluated.
This work was funded by a grant from NCRR/NIH (R24RR018827).
28 GLOBAL GENE EXPRESSION ANALYSIS OF ELONGATED EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER AND IN VITRO FERTILIZATION
Authors: Betsha S. , Salilew-Wondim D. , Havlicek V. , Besenfelder U. , Rings F. , Hoelker M. , Schellander K. , Tesfaye D.
Published: December 2011

Despite their ability to reach to the blastocyst stage, nuclear transfer embryos showed various abnormalities with respect to pregnancy outcome. We hypothesised that once cloned embryos were transferred to a suitable recipient the conceptus and endometrial interaction during peri-implantation stage was a remarkable step that determines the pregnancy outcome. In order to elucidate the molecular mechanisms underlining this phenomenon, the present experiment was conducted to compare the transcriptome profile of Day-16 elongated embryos derived from somatic cell nuclear transfer (SCNT) and fertilized controls, namely, artificially inseminated (AI) and in vitro fertilized (IVF) using bovine genome Affymetrix array. Following total RNA isolation from 3 replicates of each group, biotin-labeled cRNA was hybridized on 9 bovine chips. Data were normalized by using guanine cytosine robust multi-array analysis (GCRMA) and analysis was performed using the LIMMA package in R software. The present findings demonstrate that the gene expression profile of SCNT elongated embryos closely resembled those of the in vitro fertilized group. Only 10 genes were found to be differentially regulated between SCNT and IVF embryos (P ≤ 0.05, fold change ≥2 and false discovery rate 20%). On the other hand 303 and 336 genes were differentially expressed in IVF vs AI and NT vs AI embryos comparisons, respectively. Majority of the transcripts were found to be down-regulated on IVF embryos, whereas only 21 transcripts were up-regulated. Moreover, 158 and 178 genes were up- and down-regulated, respectively, in NT embryos as compared with AI. The NT embryos were found to be enriched with transcripts responsible for lipid metabolism (ACOX1, ACOT4 and ACOT8) and molecular transport (B4GALT1, ERBB3, MIF and PRKAG2) compared with AI. Moreover, genes involved in cell movement (CD97, CTSL1, F2R, ITGA and ITGAV) were highly abundant in IVF embryos compared with AI. Ingenuity pathway analysis of differentially regulated genes showed that metabolic and tight junction pathways and the genes involved in these pathways were activated in NT and IVP elongated embryos, respectively, as compared with AI. In conclusion, elongated embryos from NT and IVP pregnancies show differences in expression of genes involved in various biological processes compared with their AI counterparts.
34 DIFFERENTIALLY EXPRESSED PROTEINS OF EARLY-STAGE PLACENTA DERIVED FROM CLONED CAT EMBRYOS USING PROTEOMICS ANALYSIS
Authors: Bang J. I. , Bae D. W. , Kwon Y. S. , Deb G. K. , Choi B. H. , Kwon T. H. , Ha A. N. , Dey S. R. , Kim S. S. , Cho H. T. , Lee K. L. , Kong I. K.
Published: December 2011

We have previously demonstrated that differentially expressed proteins affect abnormal development and function of cloned term placenta. This is associated with cloned fetus morbidity and mortality. We also frequently observed loss of the cloned fetus and failed development during early pregnancy periods. To confirm the pattern of important gene expression in cloned placenta during pre- and post-implantation, we investigated expression pattern of proteins in early stage (21 days) domestic cat placentas of cloned embryo transfer (CEP; n = 2) and artificial insemination (CP; n = 4) derived pregnancy. The differentially expressed proteins were investigated by 2-DE and MALDI-TOF/MS. Twenty-three proteins were up- and down-regulated at least 1.5-fold in the CEP (P < 0.05) compared with the CP. Differentially expressed proteins were analysed using PDQest program and statistically analysed by 1-way ANOVA using the SPSS software. In CEP, 13 proteins were up-regulated, such as 78-kDa glucose-regulated protein (GRP78), annexin A2 (ANXA2), protein DJ-1 (DJ1), adenylate kinase isoenzyme 1 (AK1), protein disulfide-isomerase A3 (PDIA3), heat shock protein β-1 (HSPB1), actin, cytoplasmic 1 (ACTB), serum albumin (ALB), protein disulfide-isomerase A6 (PDIA6), G protein-regulated inducer of neurite outgrowth 1 (GRIN1) and triosephosphate isomerase (TIM). In contrast, 10 proteins were down-regulated, such as vinculin (VCL), triosephosphate isomerase (TIM), heterogeneous nuclear ribonucleoprotein H (hnRNPH), tropomyosin α-4 (TPM4), 60-kDa heat shock protein, mitochondrial (Hsp60), serum albumin (ALB), calumenin (CALU), keratin type 1 (CK1) and prohibitin (PHB). To validate the identified proteins in the CEP compared with the CP, we investigate a peptide sequences using MALDI-TOF/TOF tandem mass spectrometry. The sequence information obtained a high ions score from NCBI and Swiss-Prot databases. In conclusion, we did identify abnormal expression of proteins that might be associated with impaired development of CEP, which may endanger the cloned fetus during early pregnancy.
This work was partly supported by the BK21 program and the KOSEF (10525010001-05N2501-00110) and the Next-generation BioGreen21 program (No. PJ007990012011).
36 EFFECT OF DNA METHYLTRANSFERASE INHIBITOR, RG108, ON IN VITRO DEVELOPMENT AND ntES ESTABLISHMENT RATE IN CLONED MOUSE EMBRYOS
Authors: Li C. , Terashita Y. , Tokoro M. , Wakayama S. , Wakayama T.
Published: December 2011

Somatic cell nuclear transfer technique increased expectations among many for its potential to advance the regenerative therapy field. Cloned embryos, however, exhibit several epigenetic abnormalities, such as low histone acetylation or high DNA methylation levels compared with normal fertilized embryos. Therefore, increasing histone acetylation or reducing DNA methylation levels in cloned embryos using chemical treatments may improve cloning efficiency. We recently succeeded in improving the success rate of mouse cloning by using class IIb histone deacetylase inhibitors, such as trichostatin A (TSA), scriptaid and suberoylanilide hydroxamic acid. It has also been reported that 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor that is a chemical analogue of cytidine, inhibits the potential of embryos to develop into blastocysts and later to fetuses. In the present study, another DNA methyltransferase inhibitor RG108, which is thought to strongly interact with the DNMT1 active site to inhibit DNMT1 activity, was used to examine whether it could improve cloning efficiency. To determine the effects of RG108, cloned embryos were treated with 100 to 500 μM RG108. When cloned embryos were treated at the 1-cell stage (from artificial activation to 10 h, n = 219), the cloning efficiency was similar to the control group (8.2 vs 10.8%). On the other hand, when 500 μM RG108 was added to the culture medium from the 2-cell to morula/blastocyst stage (n = 113), although the developmental rate to blastocyst stage did not change significantly (79.6% vs 72.3%), higher Oct3/4 expression and more ICM cells were observed compared with non-treated, control cloned embryos. Moreover, we tried to establish ES cell lines from those cloned embryos and 11 ntES lines were generated from 21 blastocysts, which was higher than that of control (6 ntES cell lines from 20 blastocysts). All ntES lines showed AP staining positively. This finding showed that the quality of cloned mouse blastocysts increased when treated with a DNA methyltransferase inhibitor, suggesting a possible means for improving cloning efficiency in the future.
37 EFFECT OF EMBRYO STAGE ON PREGNANCY RATE FOLLOWING DIRECT TRANSFER OF BOVINE EMBRYOS FROZEN IN ETHYLENE GLYCOL
Authors: Hasler J. F.
Published: December 2011

Annually, more than 400 000 in vivo-recovered bovine embryos are officially reported by members of the Canadian and American Embryo Transfer Associations. Between 65 and 70% of these embryos are cryopreserved and more than 95% are frozen in ethylene glycol (EG). Statistics on factors affecting embryo freezing are difficult to obtain because many cattle breeders/farmers no longer report pregnancy rates back to embryo transfer (ET) practitioners. Concerns are often expressed as to the optimal stage at which to freeze bovine in vivo-derived embryos. This is a retrospective analysis of results from 5 commercial ET programs (1 in the United States, 3 in Canada and 1 in the Netherlands) for which pregnancy data relative to embryo stage at freezing were made available. Embryos representing 4 stages of development, as defined by the IETS (4 = late morula, 5 = early blastocyst, 6 = mid blastocyst and 7 = expanded blastocyst) are included in the data. The number of embryos thawed and transferred ranged from 3954 to 24 827 for the 5 programs, with a total of 72 828. Embryos were frozen in either 1.5 M EG or 1.5 M EG + 0.1 M sucrose and exposure time to cryoprotectant before cooling ranged from 4 to 40 min. Pregnancy rates are shown in Table 1. Although the pregnancy rate for stage 6 embryos was only 2.6 and 3.2 percentage points lower than stages 4 and 5, respectively, these differences were highly significant and pregnancy rates for stage 6 embryos were lower than those for stages 4 and 5 in 4 of the 5 ET programs. The small decreased survival of stage 6 embryos is probably only moderately important in a commercial context. However, the pregnancy rate of stage 7 embryos was lower than all other stages for the combined dataset as well as in all 5 ET programs, with the difference between stages 5 and 7 ranging from 6.5 to 16.4 percentage points. Clearly, stage 7 embryos survive freezing at a significantly lower rate than stages 4, 5 and 6 and neither time of exposure to EG nor inclusion of sucrose in the freezing medium provided an obvious improvement. Although bovine ET practitioners routinely attempt to collect embryos on day 7 post-oestrus, recovery of stage 7 embryos cannot always be avoided. Further investigation into factors contributing to the decreased survival of stage 7 embryos is warranted.

Table 1.Effect of embryo stage on pregnancy rate of bovine embryos frozen in EG
38 VITRIFICATION OF BOS TAURUS INDICUS AND BOS TAURUS INDICUS×BOS TAURUS TAURUS EMBRYOS PRODUCED IN THE PRESENCE OR ABSENCE OF FETAL CALF SERUM
Authors: Paschoal D. M. , Sudano M. J. , Rascado T. S. , Magalhães L. C. O. , Crocomo L. F. , Lima-Neto J. F. , Guastali M. D. , Maziero R. R. D. , Martins Jr. A. , Landim-Alvarenga F. C.
Published: December 2011

In vitro-produced Bos taurus indicus (zebu) and Bos taurus indicus × Bos taurus taurus (cross-bred) embryos behave differently when vitrified. The present experiment aimed to examine the effect of vitrification on embryos produced in the presence or absence of FCS. Cumulus-oocyte complexes (COC) were matured in TCM-199 and fertilized in human tubal fluid medium with frozen Nelore bull semen. On Day 1 (Day 0 = IVF), presumptive zygotes were cultured with SOFaa + BSA in the presence of FCS (Group 2.5%) or in the absence of FCS (Group 0%) until Day 7. The cleavage was analysed on Day 3 and the blastocyst rate on Day 7. Blastocysts were vitrified and, after warming (Campos-Chillòn et al. 2006) the viability was evaluated. Data were analysed with ANOVA, using the general linear model (GLM) of SAS (SAS Inst Inc., Cary, NC, USA). Sources of variation in the model included FCS concentration and first-order interactions; all factors were considered fixed effects. The arcsine transformation (√y/100) was applied to percentage data. If the ANOVA was significant, means were separated using the Tukey test. There was no difference in cleavage (for zebu embryos: Group 0%: 87.2 ± 6.8; Group 2.5%: 87.4 ± 9.5; for cross-bred embryos: Group 0%: 79.6 ± 11.9; Group 2.5%: 73.1 ± 13.7; P > 0.05). On the other hand, zebu embryos cultured in the presence of FCS reached blastocysts at a higher rate than cross-bred embryos in the absence of FCS (for zebu embryos: Group 0%: 33.3 ± 12.4ab; Group 2.5%: 46.8 ± 13.2a; for cross-bred embryos: Group 0%: 21.8 ± 8.3b; Group 2.5%: 33.6 ± 10.1ab; P < 0.05). After vitrification and warming, no significant differences in re-expansion rate (zebu embryos: Group 0%: 82.7 ± 13.1; Group 2.5%: 75.0 ± 9.8; cross-bred embryos: Group 0%: 93.7 ± 8.8; Group 2.5%: 84.1 ± 11.3; P > 0.05) and cell number per embryo (zebu embryos: Group 0%: 65.1 ± 34.7; Group 2.5%: 42.6 ± 17.2; cross-bred embryos: Group 0%: 64.3 ± 44.2; Group 2.5%: 52.0 ± 31.5; P > 0.05) between species groups and within species were seen. However for zebu embryos, Group 0% showed a lower damaged cell rate than Group 2.5%. The same effect was not observed in the cross-bred embryos (zebu embryos: Group 0%: 20.3 ± 22.7c; Group 2.5%: 63.3 ± 27.0d; cross-bred embryos: Group 0%: 25.4 ± 24.3cd; Group 2.5%: 45.8 ± 34.6cd; P < 0.05). The addition of 2.5% FCS had a higher deleterious effect on zebu embryos than cross-bred (zebu × taurine) embryos after vitrification. These results also reinforce the species differences observed between zebu and cross-bred, as they behaved differently in relation to the addition of FCS in the culture medium and in relation to their cryopreservation sensitivity.
Supported by FAPESP 10/50410-2.
5 PARTHENOGENETIC EMBRYONIC STEM CELLS ARE CONNECTED BY FUNCTIONAL INTERCELLULAR BRIDGES
Authors: Pennarossa G. , Tettamanti G. , Gandolfi F. , deEguileor M. , Brevini T. A. L.
Published: December 2011

We previously reported that parthenogenetic stem cells display abnormal centrosome and spindle formation that results in severe chromosome missegregation, with a high incidence of hypoploid karyotypes. Unexpectedly, this is not accompanied by a correspondingly high rate of apoptosis and, by contrast, parthenogenetic cells share the pluripotency, self-renewal and in vitro differentiation properties of their bi-parental counterparts. We hypothesise that this is possible through a series of adaptive mechanisms that include the presence of intercellular bridges similar to those that connect germ cells during spermatogenesis. This would provide a way for mutual exchange of missing cell products, thus alleviating the unbalanced chromosome distribution that would otherwise hamper normal cell functions. The presence of intercellular bridges was investigated in pig parthenogenetic embryonic stem cells (PESC) by transmission electron microscopy (TEM). Cultured cells were fixed in 2% glutaraldehyde and post-fixed in 1% osmic acid. After standard dehydration in ethanol series, samples were embedded in an Epon-Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica). Thin sections were stained and observed with a Jeol 1010 electron microscope. Pig PESC were also subjected to scanning electron microscopy (SEM). To this purpose, they were fixed and dehydrated as described above, covered with a 9-nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech) and examined with an SEM-FEG Philips XL-30 microscope. To demonstrate functional trafficking activity through intercellular canals, fluorescent 10-kDa dextran was injected into the cytoplasm of a single cell with FemtoJet Microinjector (Eppendorf). Movement of the molecule from the injected cell to others was observed with a Nikon Eclipse TE200 microscope. Ultra-structural analysis of PESC demonstrated the existence of intercellular bridges that ensured cytoplasmic continuity among cells. These canals appeared variable in size and were characterised by the presence of stabilising actin patches. Furthermore, extensive movement of 10-kDa dextran among cells demonstrated functional intercellular trafficking through these communication canals, suggesting their use for transfer of mRNA, proteins and ribosomes among cells. Our results demonstrate that PESC present a wide network of functional intercellular bridges that may constitute an adaptive mechanism to support normal cell functions. This process is commonly observed in transformed cells and gives further support to the recent hypothesis that suggests the existence of common features and links between oncogenesis and self-renewal in pluripotent cell lines.
Supported by AIRC IG 10376. PG was supported by INGM.