bluuebunny/crossref_metadata_2025 huggingface dataset:
284 HIGH-THROUGHPUT MICROFLUIDIC TECHNOLOGIES FOR STEM CELL RESEARCH
Authors: Kim D. , Monaco E. , Lima A. , Hurley W. L. , Wheeler M. B.
Published: January 2008

One of the major issues in stem cell biology is to determine the conditions that enable stem cell culture, which has been slow and laborious due to the present nature of culture systems. Our current research is designed to leverage existing robotic and fluid handling technologies with the unique fluid control and microenvironment properties of the microscale, along with the extensive expertise in stem cell research. One key advantage of microfluidic systems for stem cell research is the ability to ultra-miniaturize the cell-based assays. Swine adipose-derived stem cells (ADSCs) were cultured and differentiated into adipogenic and osteogenic cells. A fluid handling robot was used to implement the passive pumping in the microdevices, which did not require any fluid connectors. A 192-channel micro-conduit polydimethylsiloxane (PDMS) array was made using soft lithography and bonded onto a standard microplate. The fluid handling robot was programmed to load cells and change medium. The total time to change medium for 192 channels was 30 min. Swine adipose-derived stem cells were cultured using DMEM + 10% fetal bovine serum (FBS) for six days in a 100% humidified 5% CO2 atmosphere. Two different cell concentrations were compared (1 � 106 cells and 2 � 106). The ADSCs were differentiated into adipogenic and osteogenic cells using specific differentiation media for the following ten days. The medium was changed every 24 h by the fluid handling robot. To assess for the differentiation, the adipogenic and osteogenic cells were stained using oil red O and alizarin red S to verify fat and calcium formation, respectively. Fatty acid accumulation was confirmed by red-stained lipid vesicles inside the adipogenic cells, and calcium formation was observed as red-stained calcium deposits around the osteogenic cells. We also determined that the concentration of 1 � 106 cells (equivalent to 1500 cells per channel) gave better results than the concentration of 2 � 106 cells (equivalent to 3000 cells per channel) in terms of cell morphology and differentiation parameters. The use of multifactorial directed differentiation using high-speed robotic systems, as employed in our research, will enable the examination of large matrices of culture and differentiation conditions for stem cells. Furthermore, our approach enables the use of gene expression analysis and other analytical methodologies to study the differentiation and cell function of cells cultured under essentially unlimited conditions. Using the automated microscale system in large factorial experiments allows analysis of the basic mechanisms underlying stem cell development in vitro, and ultimately in vivo.
This research was supported by the Illinois Regenerative Medicine Institute (IRMI).
29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS
Authors: Feltrin C. , Lima A. S. , Monaco M. , Wilson S. M. , Kim D. , Wheeler M. B. , Rodrigues J. L.
Published: January 2008

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.
303 ESTABLISHMENT OF PREGNANCIES WITH HANDMADE CLONING PORCINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS CONTAINING AN ALZHEIMER'S DISEASE GENE
Authors: Kragh P. M. , Li J. , Du Y. , Lin L. , Schmidt M. , Boegh I. B. , Bolund L. , Nielsen A. L. , Holm I. E. , Joergensen A. L. , Vajta G.
Published: January 2008

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, specific genetic manipulations can be performed in donor cells before SCNT to derive pig models for specific human genetic diseases, including the neurodegenerative disorder Alzheimer's disease (AD). In the present study, we established pregnancies after transfer of SCNT blastocysts produced by the handmade cloning (HMC) technique. The blastocysts were transgenic for a human gene, amyloid precursor protein gene with the 'Swedish mutation' (APPsw), causing AD. For transgenesis, minipig fibroblasts were transfected by lipofection with a vector containing the APPsw gene under control of the platelet-derived growth factor β promoter (PDGF-APPsw) and a neomycin-resistance selection gene. Neomycin-resistant colonies were isolated, expanded, analyzed, and used for HMC. Cumulus–oocyte complexes were aspirated from ovaries of slaughtered sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The cytoplasts were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a 2-step fusion procedure. At the first step, 1 cytoplast was fused with 1 fibroblast in the absence of Ca2+. After 1 h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, incubated in cytochalasin B and cycloheximide for 4 h, and then cultured in PZM-3 medium. The development of reconstructed embryos to the blastocysts stage was determined after 5, 6, or 7 days of in vitro culture. To investigate the in vivo developmental capacity, blastocysts were transferred surgically to synchronized recipients. When using PDGF-APPsw-transgenic minipig fibroblasts, the rate of blastocyst formation (mean � SEM) was 39 � 3% (164/424). In comparison, non-transgenic fibroblasts resulted in a blastocyst development of 36 � 7% (36/102). In 4 recipients that received an average of 54 Day 5, 6, and 7 PDGF-APPsw-transgenic blastocysts, 2 ongoing pregnancies were confirmed by ultrasonography, 1 pregnancy was lost, and 1 returned to estrus. The results show a high in vivo developmental competence of blastocysts produced after SCNT of PDGF-APPsw-transgenic minipig fibroblasts.
308 INFLUENCE OFA MATRIX ATTACHMENT REGION ON THE EXPRESSION OF BICISTRONIC VECTORS TRANSFECTED IN MAMMALIAN CELLS CULTURED IN VITRO
Authors: Perota A. , Brunetti D. , Lizier M. , Lucchini F. , Galli C.
Published: January 2008

We investigated the effect of the 52 MAR (matrix attachment region) sequence of chicken lysozyme gene as a possible insulator for the expression of our transgenes in somatic cells to be used for nuclear transfer. With the preliminary purpose to demonstrate a possible positive effect (position or copy number) on the long-term combined expression during in vitro culture, we have created a bicistronic ubiquitous expression vector with (MAR+) or without (MAR–) MAR. The main structure of our constructs is composed of the pCAGGS promoter driving the expression of a reporter gene (enhanced green fluorescent protein, EGFP) followed by a loxable selection cassette (loxP-PGKneo). The MAR region was inserted before the selection cassette. After KpnI digestion, the resulting linearized vectors were purified and subsequently used to transfect adult porcine fibroblast cell lines using the Nucleofector system (Amaxa, Cologne, Germany). Fibroblasts were cultured in DMEM/M199 medium (1:1) + 10% FCS supplemented with 5 ng mL–1 of basic fibroblast growth factor (bFGF). In every experiment, 1 � 106 cells were transfected with 2.5 µg of linearized plasmid and selected for 3 weeks with medium supplemented with 400 µg mL–1 of Geneticin (G418 sulfate, Calbiochem, La Jolla, CA). On Day 8 of G418 selection, we analyzed 150 colonies for each treatment, using fluorescence microscopy with fluorescein isothiocyanate filters. Colonies were classified according to size (large) and cell morphology (small cells without signs of aging). In addition, colonies were classified for uniform GFP expression (uniform), patchy GFP expression (variegated), and no GFP expression (negative). Resistant colonies derived from MAR+ and MAR– vectors, respectively, had 36 (24%), 42 (28%), and 56 (37%) v. 79 (53%), 58 (39%), and 29 (19%) uniform, variegated, and negative GFP. Differences were significant for variegated and negative in MAR+ v. MAR– (chi square, P < 0.05). Thirty-six MAR+ and 42 MAR– colonies uniformly expressing GFP were transferred to 24-well plates and subjected to G418 selection until Day 22, when 7 MAR+ and 15 MAR– clones were still growing in culture. Four of seven MAR+ (57%) and 7/15 MAR– (47%) uniformly expressed high levels of GFP. In conclusion, we found that significantly fewer colonies expressed GFP with the MAR+ vector; however, within the GFP-expressing clones, expression was more uniform. Therefore, we did not find a beneficial effect of MAR sequences on expression in somatic cells during in vitro culture; however, further work is needed to investigate their effect after nuclear transfer and/or on the next generation of cloned transgenic animals.
310 PRODUCTION OF TRANSGENIC CLONED PORCINE EMBRYOS EXPRESSING EGFP OR LacZ GENE THROUGH REPLICATION DEFECTIVE RETROVIRAL VECTOR
Authors: Uhm S. J. , Gupta M. K. , Kim T. , Lee H. T.
Published: January 2008

We have demonstrated previously that retroviral-mediated gene transfer is a promising method to produce transgenic avian, porcine, and bovine embryos. This study was designed to evaluate the development potential of transgenic porcine embryos produced by somatic cell nuclear transfer (SCNT) of fetal fibroblast (pFF) cells transfected by a robust replication-defective retroviral vector harboring enhanced green fluorescent protein (EGFP) or β-galactosidase (LacZ) gene. Moloney murine leukemia virus (MoMLV)-based retroviral vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein and harboring EGFP or LacZ under the control of β-actin promoter were produced and used to transfect primary pFF cells that were subsequently used for SCNT of enucleated porcine oocytes matured in vitro. Our results showed that all surviving cells after transfection and antibiotic selection expressed the genes without any evidence of replication-competent retrovirus. The fusion, cleavage, and blastocyst rates were 85.6 � 6.5, 53.6 � 6.4, and 12.0 � 5.7% for EGFP; 83.5 � 8.2, 57.5 � 6.3 and 10.1 � 4.1% for LacZ; and 80.5 � 4.2, 60.9 � 8.2 and 12.3 � 4.0% for controls, respectively. Mosaicism was not observed in any of the group as evidenced by the expression of LacZ or EGFP in individual blastomeres of all embryos upon staining with β-galactosidase (for LacZ) or when visualized under UV illumination of an epifluorescent microscope using the fluorescein isothiocyanate (FITC) filter set (for EGFP). Further recloning of EGFP-expressing blastomeres, obtained from 4-cell-stage cloned embryos produced by SCNT of pFF cells infected with EGFP harboring vector, into enucleated metaphase II (MII) oocytes resulted in consistent expression of EGFP in recloned blastocysts. Interspecies SCNT (iSCNT) of transfected pFF into enucleated bovine oocytes could also result in consistent gene expression without any adverse effect on blastocyst rate (5.5 v. 4.9%) compared with non-transfected pFF. These data indicate that the replication-defective retroviral vector used in the present study is robust and independent of the genes inserted. Furthermore, introduction of transgenes by this method does not influence the in vitro development rate of cloned embryos.
This work was supported by a grant from Biogreen 21 Program, RDA, Republic of Korea.
314 ULTRASONOGRAPHIC ASSESSMENT OF FETAL DEVELOPMENT IN AI AND ET SHEEP FETUSES
Authors: Panarace M. , Jauregui G. , Smith J. , Ferguson D. , Hill J. , Medina M. J.
Published: January 2008

The environment to which the mammalian embryo is exposed during the preimplantation period of development has a profound effect on the physiology and viability of the conceptus (Gardner et al. 2005 Reprod. Fertil. Dev. 17, 361–370); therefore superovulation has been implicated in abnormal methylation and imprinting in the resultant embryo. In addition, the first week after mating is now known to be a critical time for embryo programming in sheep. However, the effect of altering the maternal environment by superovulation followed by embryo transfer (ET) into non-superovulated recipient ewes has not been thoroughly investigated in livestock animals. The objective of the present study was to determine if fetal and placental growth curves are affected by the mating type (AI or ET). In the ET group, 10 ewes underwent a superovulation program prior to artificial insemination; then 29 embryos were transferred singly into recipients. In the AI group, 25 ewes were inseminated with semen from the same sire as used for the ET group. All ewes used were Merino breed. At Day 30 after AI or Day 24 after ET, ewes were scanned for pregnancy using a 5-MHz transrectal probe (Honda, Tokyo, Japan). First, crown rump length (CRL, mm) was measured at Days 30 and 44, while abdominal circumference (ABD, mm) and femur length (FEM, mm) were measured monthly from Day 58 to term using a transabdominal 3-MHz convex probe. Data were analyzed by ANOVA for repeated measures. At Day 30, CRL was not different among AI and ET fetuses, but at Day 44, AI fetuses had significantly longer CRL than ET fetuses (P < 0.001, Table 1). Also, at the first measurement of FEM and ABD (Day 58) mating types were not significantly different, but measurements at Day 85 showed AI fetuses had larger FEM, P < 0.05, and ABD, P < 0.01. From Day 114 onward, there was no difference in fetal size between AI and ET fetuses. Univariate analysis of variance indicated no significant mating type or gender effects on gestation length, placenta weight, cotyledon count, weight of cotyledons as a proportion of total placenta, or weight of lambs at birth, prior to lamb-marking and weaning. We conclude that in the ET group, the first 90 days of the embryonic/fetal growth curve could subtly be affected by superovulation treatment; however, it did not affect weight and survival rate of this type of fetus.

Table 1. Mating type effects on crown rump length (CRL, mm), femur length (FEM, mm), and abdominal circumference (ABD, mm), mean ± SE
40 EARLY DEVELOPMENT OF RECONSTRUCTED MOUSE EMBRYOS USING BONE MARROW CELLS FROZEN WITHOUT CRYOPROTECTANT
Authors: Kato H. , Nakao A. , Nishiwaki M. , Anzai M. , Mitani T. , Matsumoto K. , Saeki K. , Hosoi Y. , Iritani A.
Published: January 2008

Animal cells frozen with suitable cryoprotectants have been successfully cryopreserved for long periods of time, maintaining viability upon thawing. Animal cells frozen without cryoprotectant, however, may suffer serious damage and not be useful as donors in somatic cell nuclear transfer (SCNT). However, in some cases, old animal samples were frozen only as a whole body or a piece of tissue without cryoprotectant. If the cells from such old samples could be useful for SCNT, then there are potentially many candidates where individual animals could be reproduced. In this study, we examined the possibility of using mouse bone marrow cells frozen without cryoprotectant as nuclear donors in SCNT. Thigh bones were collected from B6C3F1 mice and frozen in either a –25�C or a –80�C freezer for more than one month. Thawing of frozen bones was performed by placing them in an incubator at 37�C. Bone marrow cells were collected by washing the bone cavity with saline. Recipient oocytes for SCNT were collected from B6D2F1 female mice. The enucleation of recipient oocytes and the injection of nuclei were performed as previously reported (Wakayama et al. 1998 Nature 394, 369–374) with a piezo-actuated micromanipulator system. In this study, 4 groups of mouse cells (fresh bone marrow cells, bone marrow cells frozen at –25�C, bone marrow cells frozen at –80�C, and fresh cumulus cells) were used as the nuclear donors in SCNT. After nuclear injection, embryos were kept in mCZB medium for 1 h at 37�C. Subsequently, embryos were cultured for 3 h with 5 µg mL–1 cytochalasin B and 10 mm SrCl2 for activation and cultured for an additional 20 h in mKSOM medium. The nuclear dynamics of SCNT embryos in each donor cell group was observed using 42,6-diamidino-2-phenylindole (DAPI) staining and a fluorescent microscope at 0, 1, 7, and 24 h after nuclear injection. Data were analyzed by Student's t-test. The cell viability after thawing by trypan blue vital staining was about 20% regardless of freezing temperature. At 7 h after nuclear injection, the SCNT embryos injected with frozen bone marrow cells, regardless of freezing temperature, had more single pronuclei (67%, 54/81; P < 0.05) than SCNT embryos injected with either fresh bone marrow cells (36%, 26/73) or cumulus cells (28%, 67/236). At 24 h after nuclear injection, fewer SCNT embryos injected with bone marrow cells, either fresh or frozen, developed to the 2-cell stage (fresh: 11%, 6/56; frozen at –25�C: 21%, 5/24; frozen at –80�C: 20%, 10/49) than SCNT embryos injected with cumulus cells (58%, 185/319; P < 0.05). There was no difference in the embryonic development to the 2-cell stage among SCNT embryos injected with either fresh or frozen bone marrow cells. Further studies are required to determine whether cells frozen without cryoprotectant are capable of resulting in viable clones.
41 NUCLEAR REMODELING IS AFFECTED BY TREATMENT OF BOVINE OOCYTES WITH A PROTEASOME INHIBITOR BEFORE NUCLEAR TRANSFER
Authors: Le Bourhis D. , Gall L. , Ruffini S. , Heyman Y. , Vignon X.
Published: January 2008

Complete reprogramming of somatic cell nuclei after nuclear transfer (NT) depends on extensive remodeling of chromatin by factors present in the recipient cytoplast. M-Phase Promoting Factor (MPF) activity, responsible for nuclear remodeling in metaphase II recipients, may be lowered by oocyte enucleation and handling prior to NT. Then, a partial nuclear envelope breakdown or incomplete premature chromosome condensation (PCC) may be, in turn, associated with an inefficient reprogramming. The aim of the present study was to maintain the bovine recipient cytoplast at a high level of MPF activity during the fusion procedure by using a proteasome inhibitor, MG132, and to assess the consequences on nuclear remodeling and developmental potential. Bovine COCs were in vitro-matured for 23 h. Matured oocytes were denuded, and then incubated in TCM-199 for 45 min and enucleated in the presence (treated group) or absence (control group) of 5 µm MG132. Embryos were reconstructed by fusion with adult fibroblasts and activated in 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. In Experiment 1, MPF activity was analyzed immediately after fusion/activation by measuring the phosphorylation of exogenous histone H1, and Cyclin B expression was assessed by Western blotting. In Experiment 2, microtubules revealed by immunofluorescense with anti-tubulin antibody and chromatin stained with 10 µg mL–1 propidium iodide were analyzed by confocal microscopy 1 h after fusion/activation. In Experiment 3, NT embryos activated for 5 h were cultured in vitro for 7 days. Rate of development and cell counts in both groups were then recorded at the blastocyst stage. Remarkably, in Experiment 1, a high MPF activity was found in only 50% of the control oocytes, but MG132 treatment did not enhance this rate. On the other hand, cyclin B persisted for 2 h after activation in treated oocytes whereas it had dropped in controls. Experiment 2 revealed a higher rate of PCC in the treated embryos (n = 51) than in control embryos (n = 54): 96.0% v. 24.0% (chi-square, P < 0.001). Moreover, microtubules reorganized in a metaphasic spindle in embryos undergoing PCC, whereas cytoplasmic microtubules were observed in the others. In Experiment 3, cleavage and blastocyst rates were not significantly different between the treated (n = 92) and the control groups (n = 105): 83.7% and 53.3% v. 78.1% and 50.5%, respectively. However, the mean cell number in treated embryos (n = 27) was significantly higher than in controls (n = 20): 134 � 25 v. 109 � 43 (P < 0.05). This study suggests that MG132 treatment improved the maintenance of oocyte factors responsible for PCC in bovine NT embryos, although it did not modify MPF activity, thus questioning the role of MPF in the induction of PCC. Accordingly, PCC may be important for blastocyst quality and nuclear reprogramming in NT embryos. Full-term development of MG132-derived embryos is under investigation.
45 EFFECT OF TRICHOSTATIN A TREATMENT ON THE TERM DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER RABBIT EMBRYOS
Authors: Meng Q. , Polgar Z. , Liu J. , Dinnyes A.
Published: January 2008

The efficiency of somatic cell nuclear transfer (SCNT) is low in the rabbit. So far, there have been few live births reported and most clones died within the first 3 weeks after birth. It has been shown that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, improved cloning efficiency in cattle (Enright et al. 2003 Biol. Reprod. 69, 896–901) and mice (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). Although a recent report indicated that TSA treatment could increase the cell number of rabbit SCNT blastocysts (Xu et al. 2007 Reprod. Fertil. Dev. 19, 165), term development of TSA-treated cloned embryos in this species has not been reported. In this study we investigated the effect of TSA treatment on the term development of somatic cell nuclear transfer (SCNT) rabbit embryos. The oocytes and cumulus cells were collected from superovulated Hycole hybrid rabbits. After staining with Hoechst 33342 and locating following 1–2 s of UV illumination, the nuclei of oocytes were removed by micromanipulation, A cumulus cell was then inserted into the perivitelline space and fused with the cytoplast with three 20 μs 3.2 kV cm–1 DC pulses. Fused embryos were activated using the same electrical parameters 1 h later, treated with 2 mm 6-dimethylaminopurine and 5 μg mL–1 cycloheximide for 1 h, and then subsequently cultured in Earles Balanced Salt Solution (EBSS) with or without 5 nm TSA for 10 h. The embryos were then cultured in EBSS either overnight (before ET) or for 4.5 days. Embryos were transferred at the 2- to 4-cell stages to the recipients 22 h after collection of the oocytes from the donors. Caesarean sections were performed on Day 30 post-ET. In vitro developmental data (Table 1) showed no differences in the cleavage, blastocyst rates, and blastocyst cell numbers between the TSA-treated or untreated cloned embryos. After ET in the TSA group, one pregnant female delivered 7 live and 3 stillborn pups, but all of the live pups died within 1 h to 19 days later. In the untreated group, one pregnant female gave birth to 2 live and 1 stillborn pup. One pup died within 1 h after birth; the other survived (2.5 months old when this abstract was submitted). In conclusion, the results indicate that TSA treatment has a limited effect on in vitro development of SCNT embryos, and both TSA-treated and untreated SCNT clones can develop to term in rabbit. The effects of TSA treatment on the health of clones need further investigation.

Table 1. In vitro and in vivo development of SCNT rabbit embryos with or without TSA treatment

This study was supported by Wellcome Trust (Grant No. 070246), EU FP6 (MEXT-CT-2003-509582, MRTN-CT-2006-035468), and Chinese-Hungarian Bilateral projects (TET CHN-28/04, CHN-41/05).
48 THE AMOUNT OF TELOMERIC DNA IN LYMPHOCYTES OF DOG CLONES
Authors: Oh H. J. , Kim M. K. , Jang G. , Kim H. J. , Hong S. G. , Park J. E. , Sohn S. H. , Kang S. K. , Ryu P. D. , Lee B. C.
Published: January 2008

Controversy regarding the restoration of eroded telomere length of donor cells after the nuclear transfer process has arisen from previous studies of cloned cattle, mice, and pigs. Little is known about telomere lengths in dogs from either natural breeding or somatic cell nuclear transfer (SCNT). In this study, we investigated the amount of telomeric DNA (ATD) in the lymphocytes of growing dog clones and their somatic cell donors. One cloned male Afghan hound dog [Snuppy (Lee et al. 2005 Nature 436, 641)] and 3 cloned female Afghan hound dogs (Jang et al. 2006 Theriogenology; doi:10.1016J.THERIOGENOLOGY.2006.11.006) were obtained from somatic cell nuclear transfer (SCNT) of ear skin fibroblasts. The lymphocytes were recovered from all dog clones: their nuclear donor dogs (male donor dog, female donor dog), and six normal Afghan hound dogs (control, and 10-, 20-, 26-, 49-, 55-, and 58-month-old, respectively). The ATD was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) with a telomeric DNA probe. A telomeric probe containing the TTAGGG repeated DNA sequence was simultaneously amplified and labeled with digoxigenin (DIG) by polymerase chain reaction (PCR) using dog genomic DNA as template, a (GGGTAA)7 primer, and a DIG-labeling kit (Roche, Mannheim, Germany). To analyze the amount of telomeric DNA on the lymphocytes, at least 100 interphase nuclei were examined in each specimen. The image was captured by a digital camera (DP-70, Olympus) and analyzed using MetaMorph (Universal Imaging Co., Downingtown, PA, USA), an image analysis program. Our results indicated that the ATD in normal Afghan hounds gradually decreased with age. Although no difference in ATD was observed between 10- and 26-month-old dogs, the ATD in the 26-month-old dog was significantly higher than that in 49-, 55-, and 58-month-olds (P < 0.05). The mean percentage of telomeric DNA in Snuppy (18-month-old; 2.38%) was significantly higher than that in the nuclear donor dog (49-month-old; 2.12%) but less than that in the age-matched control (20-month-old; 2.72%; P < 0.05). The ATD in 3 female clone dogs (3-, 2-, and 2-month-olds; 3.47, 3.28, and 3.07%) were significantly higher than that in the nuclear donor (26-month-old; 2.65%). In conclusion, the mean percentages of telomeric DNA in dog clones were higher than in nuclear donor dogs, and the ATD of the cloned male dog was different from that in age-matched controls from natural reproduction. The results suggest that the amount of telomeric DNA in dog clones can be restored with the nuclear transfer of cultured donor fibroblasts, but further studies are required as to how telomere reprogramming occurs during the nuclear transfer process.
51 CHARACTERIZATION OF HISTONE H3 LYSINE 27 TRI-METHYLATION AND POLYCOMB GROUP GENE EXPRESSION IN BOVINE FERTILIZED, PARTHENOGENETIC, AND CLONED EMBRYOS
Authors: Ross P. J. , Ragina N. , Rodriguez R. M. , Siripattarapravat K. , Lopez-Corrales N. , Iager A. E. , Cibelli J. B.
Published: January 2008

The low developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos has been attributed to aberrant or incomplete epigenetic reprogramming. Tri-methylation of Histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes (Ezh2, Eed, and Suz12) and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes during bovine preimplantation development and to compare the dynamics of H3K27me3 after IVF, parthenogenetic activation (PG), and SCNT. Embryos were produced using our standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). MII oocytes and pronuclear-, 2-cell-, 4-cell-, 8-cell-, morula-, and blastocyst-stage embryos were collected for quantitative RT-PCR or immunofluorescence (IF). IF analysis of embryos was performed using primary antibodies against H3K27me3, Eed, Ezh2 (Abcam, Cambridge, MA, USA), and Suz12 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and imaged by confocal microscopy. The average background-corrected nuclear fluorescence intensity for each embryo was analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Ezh2, Eed, Suz12, and control transcript levels from oocytes and IVF-derived embryos were determined by absolute quantification as described by Bettegowda et al. (2006 Mol. Reprod. Dev. 73, 267–278). RNA levels were normalized to the external control and analyzed by ANOVA (mixed model). A steady decrease in total transcripts was observed from the MII to the 8-cell stage (P < 0.05). During this period, the levels of polyadenylated Suz12 and Eed transcripts remained steady, while Ezh2 transcripts increased at the 2-cell stage (P < 0.05) and then decreased from the 2- to 4-cell stage (P = 0.10). After embryonic genome activation, all three genes displayed an increase in transcript abundance by the blastocyst stage (P < 0.05). Concurrent IF analysis revealed that Ezh2 localized to the nucleus throughout preimplantation development, Eed localized to the cytoplasm at the 2-, 4- and 8-cell stages (expression confirmed by westernblot) and to the nucleus at morula and blastocyst stages, and Suz12 was exclusively nuclear and detected only at morula and blastocyst stages. IF analysis of H3K27me3 revealed that, for all embryo types, nuclear fluorescent intensity was highest at the MII stage, steadily decreased to reach a nadir at the 8-cell stage (P < 0.05), and then significantly increased at the blastocyst stage (P < 0.05). H3K27me3 fluorescent intensity was significantly higher in SCNT blastocysts than in IVF and PG blastocysts (P < 0.05). We conclude that H3K27me3 is efficiently removed from the embryonic and somatic cell chromatin, but is abnormally reestablished in SCNT embryos.
55 LIVE PLASMA MEMBRANE ANALYSIS OF EARLY APOPTOTIC CELL DEATH IN PORCINE ADULT DERMAL FIBROBLASTS PRIOR TO SOMATIC CELL CLONING
Authors: Skrzyszowska M. , Samiec M.
Published: January 2008

The aim of our study was to determine the in vitro developmental capability of porcine nuclear-transferred (NT) embryos reconstructed with adult dermal fibroblast cells, which had been analyzed for apoptosis by live plasma membrane fluorescent labelling. Frozen/thawed fibroblasts, which had been in vitro cultured to confluency, were used for analysis. To detect the early apoptotic changes in the plasma membrane involving the externalization of phosphatidylserine molecules and the subsequent loss of lipid composition asymmetry, the fibroblasts were tagged using a conjugate of annexinV with enhanced green fluorescent protein (eGFP). In the somatic cell cloning procedure, enucleated in vitro-matured oocytes were reconstituted with non-apoptotic dermal fibroblast cell nuclei. Afterwards, NT-derived oocytes were stimulated with a combination of electrical and chemical activation. Simultaneous fusion and electrical activation were induced by application of two successive DC pulses of 1.2 kV cm–1 for 60 �s. A two-step chemical activation procedure was initiated after a 1.5–2 h delay. The cybrids were exposed to 15 µm calcium ionomycin for 5 to 7 min and then incubated in the culture medium supplemented with 10 µg mL–1 cycloheximide for 3 h. Reconstructed embryos were in vitro cultured in NCSU-23 medium for 6–7 days. Fluorescence analysis of the adult dermal fibroblast cells revealed that a relatively high proportion of donor cells exhibited proapoptotic changes in the plasma membrane. The percentage of late apoptotic cells with advanced morphological changes did not exceed 30%. Moreover, an extremely low rate (ranging from 0 to 2%) of early apoptotic cells, with a morphologically normal, i.e., smooth (non-corrugated) and intact (non-blebbing), plasmolemma but which emitted the green eGFP-derived chemiluminescence, was detected. A total of 219 enucleated oocytes were subjected to reconstruction and 185/219 (84.5%) were successfully fused with non-apoptotic nuclear donor cells. Out of 185 cultured NT embryos, 108 (58.4%) cleaved. The frequencies of cloned embryos, that reached the morula and blastocyst stages, were 84/185 (45.4%) and 26/185 (14.0%), respectively. In conclusion, annexin V-eGFP is a sensitive method able to detect the early phases of apoptosis in cultured adult dermal fibroblast cells, because it identified that very small proportion of morphologically normal cells (without shrinkage of the plasmolemma) that also emitted the annexin V-eGFP-derived biochemiluminescence. Nonetheless, the probability of their random erroneous selection for somatic cell cloning appears to be extremely low. It was also found that the preimplantation developmental potential of NT embryos originating from non-apoptotic adult dermal fibroblast cells is relatively high.
This work was supported by the Scientific Net of Animal Reproduction Biotechnology.
59 IN VITRO DEVELOPMENT OF FELINE CLONED EMBRYOS RECONSTRUCTED WITH PREADIPOCYTES
Authors: Tomii R. , Ogawa B. , Nagashima H.
Published: January 2008

The technique of somatic cell nuclear transfer (NT) in domestic cats is expected to contribute to the conservation of wildcats, for which extinction is a concern. In this study, we examined in vitro developmental ability of cloned embryos produced using the preadipocytes of domestic cats as nuclear donors. Primary cultures of preadipocytes were established as reported previously (Yagi et al. 2004 Biochem. Biophys. Res. Commun. 321, 967–974). Briefly, fat tissue (2–3 g) was excised from an adult female cat and digested using 0.1% collagenase for 1 h at 37�C followed by centrifugation. Only mature adipocytes that were floating near the surface of the supernatant were collected and placed in a 12.5-cm2 culture flask filled with DMEM containing 20% FBS. The flask was filled with medium, tightly capped, and cultured upside down for 7–10 days, so that the floating adipocytes attached to the inner ceiling surface of the flask. When firm attachment of the cells to the ceiling surface of the flask was confirmed, the flask was then inverted and culture was continued using the routine cell culture technique for adherent cells. In vivo-matured oocytes were collected from the ovaries of domestic cats superovulated by eCG and hCG. IVM oocytes were obtained by culturing cumulus–oocyte complexes from the ovaries collected at local veterinary clinics in TCM199-based medium for 24 to 30 h. In vivo-matured and IVM oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette in the presence of 7.5 µg mL–1 cytochalasin B. The nuclei of the donor cells were transferred to enucleated in vivo-matured and IVM oocytes by means of electric fusion (300 V mm–1, 30 µs, twice). The reconstructed embryos were activated electrically (125 V mm–1, 60 µs, twice), followed by treatment with 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. The cloned embryos were cultured in vitro for 7 days in MK-1 so that their developmental ability might be examined. The fusion rate of donor cells was similar between in vivo-matured and IVM oocytes (56.8%, 21/37 v. 54.5%, 42/77). The developmental ability of NT embryos reconstructed with in vivo-matured oocytes was similar to that of NT embryos reconstructed with IVM oocytes (cleavage: 52.4%, 11/21 v. 42.9%, 18/42; development to blastocysts: 9.5%, 2/21 v. 11.9%, 5/42). The results indicate that cloned feline embryos reconstructed using preadipocytes can develop in vitro into blastocysts.
64 RECONSTRUCTION OF HETEROGENEOUS EMBRYOS BY HUMAN SOMATIC CELLS AND BOVINE ENUCLEATED OOCYTES AND ISOLATION OF PUTATIVE HUMAN EMBRYONIC STEM CELL CLONES
Authors: Zhang D. , Zhou H. M.
Published: January 2008

This study was undertaken to reconstruct heterogeneous nuclear-transferred embryos by using human fetal skin fibroblast cells as nuclear donor cells and the enucleated bovine oocytes as recipient cytoplasts for the purpose of investigating the feasibility of enucleated bovine oocyte cytoplasm as a means of reprogramming human somatic cell nuclei in an attempt to generate an accessible, autologous, and potentially unlimited source of totipotent human embryonic stem cells for transplantation medicine. Bovine ovaries were recovered at a local abattoir and oocytes were in vitro-matured and employed as recipient cytoplasts. A human fibroblast cell line was derived from an aborted fetus at 4 months of age, serum-starved, and used as donor somatic cells. The cultured nuclear transfer embryos were visually assessed for the first completion of cleavage at 48 h of culture, and for subsequent developmental stages at 72 to 168 h. The fusion of fibroblast cells into recipient cytoplasm was induced by electroporation. The fused oocytes were activated by ionomycin with 2 m mL–1 6-DMAP. The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% (v/v) fetal calf serum (FCS) for 168 h. Morulae and blastocysts were used for isolating embryonic stem cells. The results indicated that human fetal skin fibroblast cells could maintain normal morphology and characteristics in culture conditions. They proliferated constantly and presented a regular growth curve in culture. Of these cells, 83.3% retained normal numbers of chromosomes after over 20 culture passages. The skin fibroblast cells exhibited normal morphology and retained normal numbers of chromosomes (2n = 64) in serum starvation culture after undergoing freezing and thawing treatment. The first completed cleavage of xenonuclear transplantation human embryos occurred between 24 and 48 h after activation, and morula and blastocyst development was completed between 72 and 168 h. The cleavage rate and the percentage of blastocyst development of the reconstructed embryos were 80.0% and 7.5%, respectively. Putative embryonic stem cell clones were observed, with nest-like morphology, after 3–7 days of culture on a fibroblast cell layer. Identifications by alkaline phosphatase (AKP) showed that the clones presented a positive reaction, which demonstrated that the isolated stem cell clones were embryonic stem cells. This study demonstrated that xenonuclear-transferred human embryos can undergo embryonic division and subsequent development to morula and blastocyst stage, and that human fetal fibroblast nuclei can be reprogrammed in bovine enucleated oocytes. Xenonuclear-transferred human embryos can be an alternative for obtaining human embryonic stem cells.
82 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP: EFFECT ON MEIOTIC MATURATION
Authors: Moawad A. R. , Campbell K. H. S.
Published: January 2008

Oocyte cryopreservation will become an important tool for the creation of genetic resources bank in domestic animals. Many problems have been described following cryopreservation of MII oocytes, including spindle disorganization (Ledda et al. 2007 Reprod. Fertil. Dev. 19, 13–23) and loss or clumping of microtubules resulting in some scattered chromosomes (Sathananthan et al. 1988 Hum. Reprod. 3, 968–977). Freezing of immature oocytes at the geminal vesicle stage might circumvent these problems because at this stage the genetic material is contained within the contours of a nuclear envelope (Siqeira-Pyles and Landim-Alvarenga 2005 Anim. Reprod. Sci. 193, 176–182). However, the number of reports relating to cryopreservation of immature oocytes remains low and it has been suggested that immature oocytes are more susceptible to cryoinjuries (Ledda et al. 2007). The aim of the present work was to study the effect of CryoLoop (Hampton Research, Aliso Viejo, CA, USA) vitrification on survival and subsequent meiotic maturation of immature ovine oocytes. Cumulus–oocyte complexes (COCs) were isolated from ovaries of slaughtered sheep. COCs were washed three times in basal medium [BM (HEPES–TCM-199 supplemented with 10% fetal bovine serum)], and then exposed to equilibration solution (10% ethylene glycol (EG) and 0.25 m trehalose (T) in BM for 3 min. After that, COCs were exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the CryoLoop within 1 min, and immersed in liquid nitrogen. Following cryopreservation, COCs were warmed by exposure of oocytes to (1) 10% EG and 1 mT in BM, (2) 0.5 mT in BM, and (3) BM for 3 min in each solution at 39�C. Then oocytes were matured in vitro for 24 h in maturation medium, as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). The nuclear maturation of oocytes was determined using aceto-orcein staining. Oocytes were classified into four stages: germinal vesicle (GV), metaphase-I (MI), anaphase-I + telophase-I (AI/TI), and metaphase-II (MII). Oocytes at MII stage were considered matured. Data were analyzed using chi-square test. The survival rate of oocytes after vitrification and warming based on oocyte morphology was 72.6% (61/84). Numbers of oocytes remaining at the GV stage were significantly higher in the vitrified group than in the control group (43.4 v. 12.6%, respectively; P < 0.01). Maturation to MII stage was higher (63.2%, 55/87) in the control group than in the vitrified group (43.4%, 23/53); however, the difference was not significant. Also, there were no significant differences between vitrified and control groups in terms of MI and AI/TI (7.5 and 5.7 v. 10.3 and 13.8%, respectively). In conclusion, immature ovine oocytes can be vitrified using CryoLoop with high survival and maturation rates.
84 PRODUCTION OF CLONED PIGLETS FROM NUCLEAR TRANSFER EMBRYOS AFTER VITRIFICATION
Authors: Nakayama N. , Tomii R. , Ueno S. , Matsunari H. , Saito H. , Ogawa B. , Nagashima H.
Published: January 2008

Cryopreservation of cloned embryos is expected to be beneficial in improving the efficiency of somatic cell cloning in pigs. We have already demonstrated that normal piglets can be produced from in vitro-matured and fertilized (IVM/IVF) embryos vitrified at an early cleavage stage after delipation (Nagashima et al. 2007 Biol. Reprod. 76, 900–905). In this study we utilized this technique in an attempt to produce piglets from cloned embryos reconstructed with IVM oocytes. Nuclear transfer (NT) embryos were reconstructed using oocytes matured in vitro in NCSU23 and preadipocytes as nuclear donors. The embryos were cultured in PZM-5 for approximately 98 h, and those that had developed to the morula stage were delipated using a noninvasive method described previously (Esaki et al. 2004 Biol. Reprod. 71, 432–437). The embryos were treated with 4% trypsin at 38�C for 1 to 4 min to induce a slight swelling of the zona pellucida, and then centrifuged (12 000g, 38�C, 23 min) with 7.5 µg mL–1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. The embryos were cultured for 1 h and vitrified by the minimum volume cooling (MVC) method using a MVC plate (Cryotop�; Kitasato Supply Co., Tokyo, Japan) in the presence of 15% ethylene glycol, 15% DMSO, and 0.5 m sucrose as cryoprotectants. Vitrified embryos were rewarmed by immersing the MVC plate diretly into rewarming solution containing 1 m sucrose and 20% calf serum at 38�C for 1 min, followed by stepwise dilution of the cryoprotectants. The rewarmed embryos were cultured for 2 days to the blastocyst stage, and then treated with 0.5% pronase to remove the zona pellucida before transfer to the uterine horn of recipients. A total of 103 vitrified blastocysts were transferred to 2 recipient gilts. Both gilts became pregnant and farrowed 2 and 4 piglets, respectively (6/103, 5.8%). These results demonstrate that cloned piglets can be produced from NT embryos that have been cryopreserved at the morula stage using noninvasive delipation and vitrification procedures.
This study was supported by PROBRAIN.