bluuebunny/crossref_metadata_2025 huggingface dataset:
295 THE SHAPE OF PORCINE NEURAL PROGENITOR CELL CELLULAR GENEALOGIES REVEALED BY TIME-LAPSE IMAGING
Authors: Faerge I. , Egeskov-Madsen A. , Holm P.
Published: August 2011

Porcine neural progenitor cells (pNPC) derived from embryonic stem cells are capable of self-renewal and differentiation into neural and glia lineages, rendering them promising candidates for cell-based therapy of neurodegenerative diseases in a large animal biomedical model. A prerequisite for the successful future therapeutic use of pNPC is a comprehensive characterisation and understanding of the neurogenic process. This is important for learning how to direct cell fates into required proportions of the cell type wanted for the specific brain disease to be treated, and it is crucial for avoiding uncontrolled cell proliferation leading to fatal tumour formations. Time-lapse analysis is a powerful tool to obtain live cell characterisation by analysing individual cell fate. Information on cellular development, division, and differentiation can be composed into a pedigree-like structure denoted as cellular genealogy giving an overview of the proliferation profile of a cell culture and the duration of each cell cycle (Al-Kofani et al. 2006). The aim of the study was to construct cellular genealogies of pNPC and differentiated neural lineages, respectively, by time-lapse imaging to evaluate the effect of external variables observed by changes in the topology of the cellular genealogy. Porcine NPC were derived from epiblast cells isolated from day-9 porcine blastocysts and cultured in DMEM/12, Pen/strep, B27 and N2 with basic fibroblast growth factor and epidermal growth factor, and differentiation was obtained by withdrawal of basic fibroblast growth factor and epidermal growth factor. The state of cellular development of undifferentiated and differentiated pNPC was verified immunohistochemically by the presence of SOX2, NESTIN, TUJI, and GFAB (Rasmussen et al. 2010). The time-lapse images were captured by a Nikon Biostation with a 10× resolution under phase contrast in a humidified chamber at 38°C with 5% CO2, 5% O2, and 90% N2. For each sequence, images were captured at intervals of 10 min in 16 frames. Sequences 1, 2, and 3 constituted passage 15 pNPC, passage 4 pNPC, and presumably differentiated cells, respectively. For each sequence, cell cycle length was calculated after manual tracking of selected cells. The cell cycle length of pNPC is shown in Table 1. Based on these data, cellular genealogies characteristic of each individual cell type have been constructed.

Table 1.Cell cycle length of porcine neutral progenitor cells (pNPC) before and after differentiation
30 SCRIPTAID TREATMENT IMPROVES POST-IMPLANTATION DEVELOPMENT OF SHEEP CLONED EMBRYOS
Authors: Bordignon V. , Albornoz M. , Colato C. , El-Beyrouthi N. , Mellano J. I. , Meltsas A. , Mellano F. , Mellano P. H. , Mellano M. L. , Mellano M. A. , Mellano J. C. , Baldassarre H.
Published: August 2011

Increased histone acetylation by exposure to inhibitors of deacetylase enzymes has been reported to improve development of embryos produced by somatic cell nuclear transfer (SCNT). However, the response to such treatment seems to vary according to the species, cell line, and type of inhibitor used. The main objective of this study was to evaluate if treatment with the histone deacetylase inhibitor Scriptaid could improve the development to term of sheep SCNT-embryos. The 2 fibroblast cell lines used in this study were obtained from skin biopsies collected from 2 adult rams of the Santa Ines breed. Oocytes were collected by laparoscopic ovum pickup (LOPU) from 30 crossbred sheep that were hormonally stimulated as described previously (Baldassarre et al. 2002 Theriogenology 57, 275). Oocyte maturation, cell transfer, fusion and activation, culture and transfer to recipients were conducted following procedures previously described (Baldassarre et al. 2003 Cloning Stem Cells 5, 279). Briefly, oocytes were matured in vitro for 24 h in TCM 199 supplemented with hormones and 10% fetal bovine serum, at 38.5°C in 5% CO2. Cells were transferred into enucleated oocytes, followed by electric fusion using a single DC pulse of 1.6 kV cm–1 for 70 μs. The reconstructed embryos were then activated using ionomycin (5 μM/5 min) followed by cycloheximide (10 μg mL–1) and cytochalasin B (7.5 μg mL–1) for 4 to 5 h and then cultured in mSOF media (control); while half of the reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Subsequent to culture in mSOF ± Scriptaid as above, selected embryos were finally transferred into the oviducts of synchronized recipients within 24 h from fusion. Pregnancy was detected and monitored for the first 3 months by transrectal ultrasound scanning. A total of 258 oocytes were recovered (8.6/donor), of which 203 resulted in fused embryos after micromanipulation (79%) and 178 (69%) were selected for transfer into the oviducts of 18 synchronized recipients (avg. 10 embryos/recipient). Initial pregnancy was significantly higher in the Scriptaid group (40 v. 12.5%; P < 0.01). Interestingly, pregnancy was maintained through gestation Day 90 in the Scriptaid group, while the pregnant recipient carrying the control embryos lost her pregnancy by Day 60. All 4 pregnant recipients are due in early August. Our results are consistent with a previous report from (Zhao et al. 2010 Cel. Reprog. 12, 75) working with pig embryos and suggest that Scriptaid treatment can improve post-implantation development of SCNT sheep embryos. The results above will be further evaluated when data from births becomes available.
301 IMPRINTED GENE EXPRESSION PATTERNS IN CULTURED PRIMORDIAL GERM CELLS DERIVED FROM PORCINE EMBRYOS BETWEEN DAY 25 AND 30
Authors: Lee C. K. , Park C. H. , Uh K. J. , Park J. K. , Kim H. S.
Published: August 2011

Although epigenetic reprogramming during germ cell development has extensively been studied in the mouse, little is known about the timing of imprinting accomplishment in the pig. Therefore, the aim of this study was to investigate the timing of epigenetic reprogramming in porcine primordial germ cells (PGC) through short-term culture as well as to confirm the differences of epigenetic features between PGC and embryonic germ cells (EGC). The present study aimed to investigate mRNA expression pattern of imprinted genes in cultured PGC that were derived from embryos at Day 25 to 30 of pregnancy. The overall expression pattern can be separated into 3 different features: no changes in all PGC regardless of embryonic day, gradual increases through embryonic day, and extreme increases in PGC derived from embryos at Day 30. Of 8 imprinted genes, Grb10, Peg10, and Snrpn transcripts were retained through all stages. The expression level of H19, Igf2, Igf2r, and Peg1/Mest progressively increased in PGC derived at later stages of embryo development. Interestingly, Xist and Nnat transcripts showed extremely high levels of expression in PGC derived from embryo at Day 30 of pregnancy. The expression level of these transcripts was 3- to 4-fold higher in PGC derived from embryo at Day 30 of pregnancy compared with that of the others. Our results indicated that analysis of the imprinted gene expression level in short-term cultured PGC may in part reflect epigenetic modifications during germ cell development in pig. This study could help us understand how epigenetic reprogramming is maintained or lost during germ cell development. Further study will be carried out to compare imprinted gene expression patterns between the porcine PGC and EGC.

This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).
302 IMPRINTED microRNA ARE DIFFERENTIALLY EXPRESSED IN ADULT MOUSE TESTES-DERIVED MALE GERM-LINE STEM CELLS
Authors: Shin J. Y. , Jung Y. H. , Gupta M. K. , Uhm S. J. , Shin S. T. , Han Y. M. , Lee H. T.
Published: August 2011

Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells, can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which, upon testicular transplantation, produce teratomas instead of initiating spermatogenesis. This study evaluated the DNA methylation and expression of imprinted microRNA (miRNA) in mouse GS and maGS cells. The GS and maGS cell lines were established essentially as described earlier (Jung et al. 2010 Mol. Hum. Reprod. PMID: 20610616) and were quantified for maternally (miR-296-3p, miR-296-5p, miR-483) and paternally (miR-127, miR-127-5p) imprinted miRNA by real-time TaqMan® MicroRNA assay and for DNA methylation at imprinting control regions of respective miRNA (Gnas-Nespas DMR, Igf2-H19 ICR, and Dlk1-Dio3 IG-DMR) by bisulfite genomic sequencing. Sperm and embryonic stem (ES) cells were used as controls for comparison. Results showed that, similar to sperm, expression of maternally imprinted miRNA was consistently higher (P < 0.001), whereas that of paternally imprinted miRNA was consistently lower (P < 0.001) in GS cells than in control ES cells. The DNA methylation analyses further confirmed that imprinted miRNA were androgenetic in GS cells. On the other hand, DNA methylation of maGS cells resembled that of ES cells, but the expression pattern of imprinted miRNA was intermediate between that of GS cells and ES cells. The expression of imprinted miRNA in GS and maGS cells was also altered during their in vitro differentiation but varied with both the differentiation stage and the miRNA. In conclusion, our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNA which changes to an ES cell-like pattern upon their conversion to maGS cells and, therefore, may serve as an epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.

This work was supported by a grant (Code #200901FHT010305191) from BioGreen 21 Program, RDA, Republic of Korea.
303 IN VITRO NEURONAL DIFFERENTIATION OF MESENCHYMAL STEM CELLS DERIVED FROM CANINE EAR SKIN
Authors: Lee J. H. , Lee Y. M. , Maeng G. H. , Lee S. L. , Rho G. J.
Published: August 2011

The canine has been a useful animal model for the study of fundamental mechanisms and the testing of new therapies for several human pathologies using mesenchymal stem cells (MSC). For preclinical applications, the most commonly used source of canine MSC is bone marrow. Because the amount of autologous bone marrow that can be obtained is limited, skin tissue could supply a noninvasive alternative with large quantities available for the establishment of MSC. In this study, we isolated canine MSC (cMSC) from ear skin and evaluated the expression of transcriptional factors and in vitro differentiation into multiple mesenchymal lineages. The cMSC isolated from the ear skin of a female beagle dog (6 years old) were cultured in advanced-DMEM/F12 (1:1, v/v) supplemented with 10% serum replacement at 37°C, 5% CO2 in a humidified atmosphere. The cMSC at passage 3 were analysed for expression of surface markers (CD44, CD90, and CD105) and transcriptional factors (Oct-4, Nanog, and Sox2) using flow cytometry, immunohistochemical staining and RT-PCR, respectively. Differentiations into adipocytes and osteocytes of cMSC were carried out under controlled conditions for 2 and 4 weeks and evaluated by staining (Oil Red O, von Kossa and Alizarin Red S, respectively). The cMSC were induced to differentiate into neural cells in the controlled condition for 6 h. Neuronal differentiated cMSC were evaluated by immunohistochemical staining, RT-PCR, and Western blot of specific markers of neuron, such as Î2-tubulin, microtubule associated protein (MAP-2), neuronfilament M (NF-M), nerve growth factor (NGF), and nestin. The MSC surface markers such as CD44, 90, and 105 were highly detected, and transcriptional factors (Oct-4, Nanog, and Sox2) were expressed in cMSC. Adipocyte induced cells were positive by staining with Oil Red O, and osteocytes were stained by von Kossa and Alizarin Red S. Neuronal specific markers such as Î2-tubulin, MAP-2, NF-M, NGF, and nestin were expressed in the neuron induced cMSC. In conclusion, canine ear-skin-derived MSC have the capacity for differentiation into multiple lineages and have a confirmed great capability for neuronal differentiation. Hence, canine ear skin tissue could be considered a source for applications of MSC for neuronal regeneration therapy of canine and a preclinical research model for human.

This work was supported by Grant No. 2007031034040 from Bio-organ and Grant No. 200908FHT010204005 from Biogreen21.
309 DEVELOPMENT OF A CULTURE SYSTEM CAPABLE OF LONG-TERM MAINTENANCE OF BUFFALO (BUBALUS BUBALIS) EMBRYONIC STEM CELLS
Authors: Sharma R. , George A. , Kamble N. M. , Singh K. P. , Panda S. K. , Chauhan M. S. , Singla S. K. , Manik R. S. , Palta P.
Published: August 2011

The present study was aimed at developing a system for long-term culture of buffalo embryonic stem (ES) cells, which, to our knowledge, have not been maintained beyond passage 10 in reports available to date, primarily because of lack of information on their specific requirements during in vitro culture. Inner cell mass (n = 181) cells, mechanically isolated from in vitro produced day 8 blastocysts, were cultured on mitomycin-C-treated buffalo fetal fibroblast feeder layers in stem cell medium (SCM), which consisted of Knockout-DMEM® + 15% Knockout serum replacer® + 1% minimal essential medium nonessential amino acids + 50 μg mL–1 of gentamicin, supplemented with 1000 IU mL–1 of leukemia inhibitory factor (LIF) and fibroblast growth factor-2 (FGF-2) at different concentrations. The medium was changed every 24 h. The primary colony formation rate, which was similar for 5, 10, 20, and 40 ng mL–1 of FGF-2 (63.7 ± 5.2, 65.7 ± 6.5, 57.0 ± 10.5, and 62.8 ± 13.30, respectively), was significantly higher (P ≤ 0.05) than that of controls (22.4 ± 5.5). In Experiment 2, ES-cell-like cell colonies at passages 6 through 7 (n = 441) were cultured for 5 to 6 days to examine the effects of media supplements. The percentage of colonies that survived was significantly higher (P ≤ 0.05) when these were cultured in SCM+LIF+5 ng mL–1 of FGF-2 (93.1 ± 1.8) than when these were cultured in SCM alone (73.5 ± 9.0) or in SCM supplemented with FGF-2 (88.8 ± 5.4) or LIF (85.8 ± 3.7). Following examination of the colony size at 0 and 120 h of culture, the increase in colony size was found to be nearly 4- (P ≤ 0.01) and 2-fold higher (P ≤ 0.05) with SCM+LIF+5 ng mL–1 of FGF-2 (41.9 ± 3.4) and SCM+FGF-2 (21.0 ± 3.0), respectively, than with SCM alone (10.8 ± 2.6) or with SCM+LIF (9.3 ± 3.3). The ES cell colonies cultured in the presence of FGF-2 were compact and had defined edges, whereas those cultured in its absence were less compact, irregularly shaped, and had less defined edges. To confirm the role of FGF-2 in maintenance of buffalo ES cells, the cell colonies cultured in the presence of 5 ng mL–1 of FGF-2 (n = 487) were exposed to different concentrations (10, 20, or 30 μM) of SU5402, a FGF-2 receptor inhibitor, for 5 to 6 days. The percentage of cell colonies that were found to have differentiated was significantly higher (P ≤ 0.05) when these had been cultured in the presence of 30 (78.6 ± 4.2) or 20 μM (47.9 ± 1.0) than when these were cultured with 10 (24.5 ± 5.1) or 0 μM (28.6 ± 2.3) of SU5402. Following culture in SCM+LIF+FGF-2, buffalo ES cells, in which the expression of pluripotency markers such as OCT-4, NANOG, and SOX-2 was regularly confirmed, have been maintained for more than 80 passages for over an year’s time to date, indicating that a combination of LIF and FGF-2 is beneficial for the maintenance of buffalo ES cells.

Supported by NAIP grant No. C4/C-2067 from ICAR, India.
319 SUPEROVULATORY RESPONSE IN JAPANESE BLACK CATTLE BY A SINGLE SUBCUTANEOUS ADMINISTRATION OF PURE FOLLICLE-STIMULATING HORMONE DISSOLVED IN SALINE
Authors: Hiraizumi S. , Nishimoto N. , Ishiyama O. , Nishinomiya H. , Oikawa T. , Sano F. , Sakagami N. , Yamamoto M. , Nishino O. , Ooishi K. , Kurahara T. , Hashiyada Y.
Published: August 2011

The objective of this study was to evaluate the superovulatory response to a single SC administration of pFSH (Antrin R-10) dissolved in saline in Japanese Black cattle. Forty Japanese Black cows were divided into 5 groups. All cows received a progesterone releasing intravaginal device (PRID) at a random stage of the oestrous cycle except the day of oestrus (Day 0). Superovulatory treatments were initiated on Day 4 with single SC administration of 20 AU pFSH dissolved in 10 mL of saline (Group A) or 50 mL of saline (Group B), or 30 AU pFSH dissolved in 10 mL of saline (Group C) or 50 mL of saline (Group D). Conventional superovulatory treatment (Group E) was also initiated on Day 4 with 20 AU pFSH in decreasing doses in IM administration twice a day for 3 days (5/5, 3/3, and 2/2 in the am/pm). Each administration was carried out in the neck region. For oestrus induction, all animals were treated with prostaglandin F2α (750 μg of cloprostenol) on Day 4, and then the PRID was removed on Day 6 in the morning. The GnRH (10 μg of buserelin) was administrated at 31 h after PRID removal to induce ovulations. The cows were artificially inseminated at 24 h after GnRH treatment, and embryos were recovered 7 days after the insemination. Blood samples were collected at 0, 2, 4, 6, 8, 10, 24, 31, 48, 55, 72, and 79 h after the first pFSH administration, and serum pFSH concentration was measured using time-resolved fluorescence immunoassay. Percentage data were arcsine transformed before analysis, and one-way ANOVA was used for statistical analysis (P < 0.05). There were no significant (P > 0.05) differences among groups in the number of corpus luteum, total number of ova/embryos, number of transferable embryos, and percentage of transferable embryos. The concentrations of pFSH in serum increased in all groups 2 h after each administration and reached a peak at 8 h in Groups A and C, at 10 h in Groups B, D, and E, and then in all groups it decreased gradually. The concentrations of pFSH in serum decreased to the base level (the level at 0 h) in Group A at 55 h and in Group C at 72 h after the first pFSH administration. At 79 h, serum pFSH was still detected in Groups B, D, and E. These results suggest that the single SC administration of 20 AU pFSH that is the same dose as the conventional treatment is able to induce a superovulatory response comparable to that of conventional treatment in Japanese Black cattle.

Table 1.Superovulatory responses (mean ± SE) in different treatment groups
32 A CLONED FOAL PRODUCED USING OOCYTES RECOVERED BY TRANSVAGINAL ASPIRATION OF IMMATURE FOLLICLES
Authors: Choi Y. H. , Norris J. D. , Velez I. C. , Jacobson C. C. , Hartman D. L. , Hinrichs K.
Published: August 2011

Closure of all the horse slaughterhouses in the US has reduced the availability of equine oocytes in this country. We investigated the use of oocytes collected from immature follicles of live mares for cloning research. Because blastocyst development of equine cloned embryos is typically low (<10%), we also investigated the effect of Scriptaid, a histone deacetylase inhibitor that increases blastocyst development, live birth rate, and neonatal health in cloned mice and pigs. Immature oocytes were transvaginally aspirated from all follicles ≥8 mm diameter in a herd of 11 mares. The oocytes were cultured in modified M199 for 24 to 26 h. Donor fibroblasts from a 27-year old stallion were treated with roscovitine for 24 h, then were directly injected into enucleated oocytes using the Piezo drill. Reconstructed oocytes were activated with ionomycin followed by injection of sperm extract and culture with 6- dimethylaminopurine (6-DMAP) for 4 h. Recombined oocytes in the Scriptaid treatment were cultured in the presence of Scriptaid, 250 nM, starting at the onset of 6-DMAP treatment and continuing for a total of 18 to 20 h. After embryo culture, blastocysts were shipped for transfer to recipient mares. Overall, each oocyte donor mare underwent aspiration up to 10 times; 653 follicles were aspirated and 271 oocytes were recovered. The in vitro maturation rate was 65% (172/263). After nuclear transfer procedures, 147 oocytes survived; 130 were used for the study. The blastocyst development rate was 2/47 (4%) in the control treatment and 1/83 (1%) in the Scriptaid treatment. All 3 blastocysts yielded pregnancies after transfer. Both control pregnancies were lost, 1 at 30 days and other at 9 months. The mare pregnant with the embryo from the Scriptaid treatment foaled at 326 days of gestation. The foal had medical issues at birth similar to those seen in some cloned foals previously, including maladjustment, patent urachus, and poor oxygenation. These issues were resolved with medical care; the foal is 3 months of age and healthy at the time of writing. These results indicate that immature oocytes obtained from a limited number of mares can be used successfully for nuclear transfer, providing the opportunity to control the mitochondrial identity of the host cytoplast. Scriptaid treatment did not improve the rate of blastocyst development or prevent health problems at birth; however, transfer of 1 embryo in this treatment produced a viable foal. More work is needed to determine the effect of histone deacetylase treatment on efficiency of cloning in the horse.

This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts. We thank Drs. Malgorzata Pozor, Margo Macpherson, and the Medicine team at the University of Florida for medical care of the foal.
324 NEGATIVE SELECTION DOES NOT FURTHER INCREASE THE EFFICIENCY OF A PROMOTER TRAP
Authors: Beaton B. P. , Wells K. D.
Published: August 2011

Every year more than 100 000 people need organ transplants and in 2009 there were only 20 139 deceased organ donors. Genetically modified swine may be able to fill this unmet clinical need. It is clear that multiple transgenes will be required to make porcine organs or cells compatible with human recipients. Efficiencies in production of xenotransplantation pigs could be gained if multiple transgenes could be sequentially stacked at a single locus. The larger purpose of this project is to evaluate a site-specific recombination system as a tool to sequentially add multiple transgenes to the porcine α galactosyltransferase (α1,3-Gal) locus. One xenotransplantion related transgene, human decay accelerating factor (hDAF), is likely to be a part of any successful project because hDAF can prevent acute rejection by deactivating the complement system. To generate transgenic pigs that express an hDAF transgene that is genetically linked to an α1,3-Gal disruption, an α1,3-gal gene targeting vector was constructed with homologous arms that were 4.8 kb (5′ arm) and 1.8 kb (3′ arm) and were derived from genomic sequence from intron 8 and exon 9 of the α-Gal locus. Within exon 9, an IRES-Neo cassette and a CAG-hDAF cassette were inserted. A Phi-C31 AttB site was also included in the construct to later receive additional transgenes via a site-specific recombination system. This vector (pBB7) is designed to utilise a promoter-trap strategy as a method of enrichment for targeting events. We hypothesised that further enrichment could be obtained by the addition of a negative selectable marker. The goal of the experiment presented here was to evaluate this hypothesis by adding a truncated diphtheria toxin cassette (TdT) to pBB7 as a negative selection directed against random integration events. Two additional plasmids were constructed that harbored TdT, pBB8.1 (single TdT addition) and pBB8.2 (addition of 2 TdT cassettes). Four treatments (Trt) were performed in triplicate: Trt1, pBB7; Trt2, pBB8.1; Trt3, pBB8.2; and Trt4, pBB7 co-transfected with TdT. Targeting efficiencies were 13.9, 11.1, 5.6, and 6.1%, respectively. Successful targeting was assessed by PCR with primers specific for a targeting event. The first pair of primers flanked the 5′ arm of the construct and the neomycin resistance gene cassette. The second pair of primers flanked the 3′ arm of the construct and hDAF gene cassette. Although all treatments produced targeting events at very high rates, TdT inclusion in the vector (1 or 2 copies) or as a co-transfectant did not further improve the promoter trap strategy. It is concluded that a negative selectable marker may not further improve the efficiencies provided by a promoter trap strategy.
328 ANALYSIS OF FLUOROPHORE-EXPRESSING SPERMATOZOA FROM TRANSGENIC BOARS PRODUCED BY SLEEPING BEAUTY TRANSPOSITION
Authors: Garrels W. , Holler S. , Struckmann C. , Taylor U. , Ehling C. , Rath D. , Niemann H. , Ivics Z. , Kues W. A.
Published: August 2011

The pig is an important model for biomedical research. Recently, we described a method for producing transgenic pigs using a nonautonomous Sleeping Beauty (SB) transposon1 (Garrels et al. 2010 Reprod. Domest. Anim. 45, 65 abst.). Briefly, in vivo developed porcine zygotes were co-injected with a CAGGS-Venus transposon and hyperactive SB100. A total of 141 in vivo developed zygotes were injected and transferred to synchronized foster sows. Subsequent analysis revealed specific transposase-mediated integration of 1 to 5 copies of the Venus transposon in fetuses and piglets. This method results in highly efficient SB-mediated transgene transposition into the porcine genome: 57% of the fetuses examined and 42% of piglets were transgenic, representing 6.4% of the treated zygotes. The piglets showed persistent expression of the Venus reporter. Here, we present cellular analysis of 2 founder boars. Expression of the Venus reporter was observed in skin, cultured fibroblasts, leukocytes, and spermatozoa of both animals. However, flow cytometric measurement of leukocytes and cultured ear fibroblasts revealed that these boars carried both a Venus-fluorescence-positive population and a Venus-fluorescence-negative cell population. PCR analysis revealed that the Venus-fluorescence-negative cells were genotypically negative, indicating transgene mosaicism. Interestingly, all spermatozoa tested were Venus-positive and gave a distinct fluorescence peak in repeated flow-cytometric measurements (n = 6). Fluorescence microscopy revealed localisation of the Venus fluorophore in the sperm tail, in the midpiece, and in the equatorial segment of the sperm head. Motility of the transgenic sperm as measured by computer-assisted sperm analysis (Hamilton-Thorne, Beverly, MA, USA) indicated no decrease in percentage of motile sperm and the movement patterns. Sorting Hoechst 33342–stained transgenic sperm into X- and Y-chromosome bearing populations did not reveal any differences in Venus fluorescence between these 2 groups. To test the fertility of the transgenic sperm, 6 wild-type sows were artificially inseminated. Four pregnancies were established, 2 of these sows were sacrificed on Day 29 of gestation and a total of 9 Venus-positive normally developed fetuses and 2 degenerated fetuses were recovered. The other 2 pregnancies are ongoing at the time of writing. This is the first characterisation of spermatozoa from transposon transgenic pigs. The results show that Venus transposon–bearing transgenic spermatozoa are fertile and demonstrate germline transmission to the F1 offspring. Sleeping Beauty-mediated transposition is thus a promising approach for genetic modification of the pig genome.
329 GENETIC TARGETING OF THE PORCINE α1,3-GALACTOSYLTRANSFERASE GENE IN FETAL FIBROBLAST CELLS USING ZINC FINGER NUCLEASES
Authors: Hauschild J. , Queisser A. L. , Carnwath J. W. , Cost G. , Santiago Y. , Rebar E. , Gregory P. , Niemann H. , Petersen B.
Published: August 2011

Hyperacute rejection after porcine-to-human xenotransplantation is caused by binding of preformed human antibodies against Gal-epitopes on the surface of porcine cells. Organs from Gal-negative pigs have shown prolonged survival after transplantation into baboons. Knocking out a gene by conventional gene targeting frequency is extremely inefficient (homologous recombination = 0.0001 to 0.001%; Denning et al. 2001). Recent publications in rats (Geurts et al. 2009) show that the gene knockout via zinc finger nuclease (ZFN)-driven nonhomologous end joining (NHEJ) can be enhanced 10 000-fold over conventional approaches, making it feasible to generate a biallelic gene knockout with one ZFN application. Here, we used ZFN technology to generate porcine cells that carry a ZFN-mediated knockout of the Gal gene to use these cells as donor cells in somatic cell nuclear transfer (SCNT) to obtain live offspring. One primary porcine fetal fibroblast cell line was transfected by electroporation (n = 6) with a pair of ZFN plasmids designed to target the DNA sequence encoding the catalytic domain located in exon 9 of the α1,3-gal locus. Transfected cells were incubated (7 days at a combination of 30°C and 37°C) and analysed for Gal expression by fluorescence activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. On average, 1.4% (± 0.3%; n = 6) of the cells were free of Gal epitopes, indicating a biallelic knockout. DNA mutation detection analysis (Cel-I assay) of cell cultures gave a mean frequency of 3.5% NHEJ (± 1.3%; n = 6) giving the fraction of mutant alleles within the cell population. One cell line with 1% Gal-negative cells was sorted by a magnetic Dynabead-based separation method to select for Gal-negative cells (Fujimura et al. 2008). Because of the limited amount of Gal-negative cells within the cell population, we chose to select the cells with magnetic beads. This method is gentler to the cells and leads to a higher plating efficiency after sorting compared with FACS. The sorted cells could be easily expanded and will serve as donor cells in SCNT to show the feasibility of generating knockout pigs via ZFN-mediated gene knockout. This study demonstrates that ZFN technology is an applicable tool to produce genetically modified porcine cells for use as donors in SCNT and to speed the creation of pig models for xenotransplantation and human diseases.
330 REDUCED HYPERACUTE REJECTION BY TRIPLE TRANSGENIC EXPRESSION OF HUMAN COMPLEMENT REGULATORY FACTORS (hDAF and hCD59) AND H-TRANSFERASE
Authors: Jeong Y. H. , Jang G. H. , Hwang I. S. , Park C. H. , Lee H. J. , Jeong Y. W. , Hyun S. H. , Kim Y. W. , Shin T. , Jeung E.-B. , Hwang W. S.
Published: August 2011

The present study was conducted to establish a porcine transgenic cell line with human CRPs and HT genes, focused on hyperacute rejection (HAR) considering clinical xenotransplantation as alternative sources of human organs. As a first step towards establishing the stable cell line, the cDNA for 3 genes encoding human DAF, CD59, and H-transferase were cloned and sequenced. A tricistronic expression vector was constructed with the aid of 2 IRES elements (pCMV-hDAF_IRES-hHT_IRES-hCD59). The CMV-based expression vector was then introduced into miniature pig ear fibroblast cells by electroporation. Reverse transcription PCR analysis revealed that cell lines stably expressing human transgene-specific transcripts were established. The inhibitory effect of immune response in the established transgenic cell lines was measured by human serum-mediated cytolysis assay, as measured by ELISA. Under the assay conditions (based on human serum from 10 to 50%), the transgenic cell group showed significantly greater survival rate under various serum concentrations than did the nontransgenic cell control group. Moreover, the transgenic cell lines used as nuclear donors for a subsequent NT experiment were confirmed to be expressing their transgene transcripts in vitro developed preimplantation stage embryos. These results indicated that the established cell lines with human transgenes might have an inhibitory effect against lysis by human complement. It is possible that these transgenic cells could serve as nuclear donors to produce transgenic cloned pigs for xenotransplantation.
334 CONSTRUCTION OF A GENETICALLY ENGINEERED PIG EXPRESSING GREEN FLUORESCENT PROTEIN (GFP)-LABELLED PROTEASOMES
Authors: O'Gorman C. W. , Zhao J. , Samuel M. S. , Walters E. M. , Prather R. S. , Sutovsky P. , Sutovsky M. , Wells K. D.
Published: August 2011

Proteasomes are large protein complexes involved in protein degradation in eukaryotes and undergo dynamic redistribution between cellular compartments. Characterising the cellular localization of proteasomes at various stages of development and in response to stimuli is of interest. We hypothesised that porcine proteasomes could be visualised in vivo via a ubiquitously expressed transgene fusion comprising a proteasomal subunit and green florescent protein (GFP). The full-length sequence for porcine PSMA-1 was first constructed in silico from public data and was used to retrieve a GenBank expressed sequence tag (EST) sequence that appeared to be full length (accession CO946059; kind gift from R. S. Prather). Primers were designed to remove the stop codon and create homology for cloning with InFusion (Clontech, Palo Alto, CA, USA). The amplimer was inserted into pCAG-CreGFP (Addgene plasmid 13776) in place of the Cre coding region. The resulting plasmid (pKW14) was screened via restriction digest and sequenced for confirmation. This plasmid was confirmed functional in porcine fetal fibroblasts. After removal of the plasmid backbones, pKW14, a G418 resistance cassette (NEO), and the chicken egg white matrix attachment region were co-electroporated into male fetal fibroblasts (10 μg of total DNA, 5:2:2 ratio, respectively). Cells were grown in DMEM with 10% fetal bovine serum (FBS) and selection was initiated 36 h after transfection. Following 12 days of selection at 400 mg L–1 G418, colonies were screened by epifluorescence. Positive colonies were harvested and confirmed transgenic for all 3 input DNAs. Positive colonies were randomly pooled as sets of 3 independent integration events. Embryos were reconstructed via SCNT and transferred to 2 recipients. The fusion rates were 70 and 78%, respectively, with transfer numbers of 120 and 125 fused couplets being transferred into synchronized recipients on Day 0 of heat. Both recipients became pregnant and delivered 2 piglets each on Day 114 by Caesarean section. One live piglet was produced from each litter. Of the 2 live-born piglets, 1 survived beyond Day 3 and continues to be healthy. Transgenic status was verified by PCR. Expression was confirmed by epifluorescence of GFP-labelled proteasomes. This founder will be used to establish a model to evaluated cellular localization of proteasomes in vivo and in culture.
38 DNA METHYLATION IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER
Authors: Deshmukh R. S. , Oestrup O. , Oestrup E. , Vejlsted M. , Niemann H. , Lucas-Hahn A. , Petersen B. , Li J. , Callesen H. , Hyttel P.
Published: August 2011

DNA de- and re-methylation are crucial for reprogramming of the differentiated parental/somatic genome in the ooplasm. The presented research was aimed at analysis of the DNA methylation dynamics in porcine preimplantation embryos developed in vivo (IV) and produced in vitro by IVF, somatic cell nuclear transfer (SCNT), and parthenogenetic activation (PA). Embryos of early and late 1-cell, 2-, 4-, and 8-cell, and early and late blastocysts stages obtained by the mentioned methods were fixed in 4% paraformaldehyde and subjected to immunocytochemistry using anti-5MetC (Mouse monoclonal, Abcam, Cambridge, MA, USA) antibody. DNA was labelled using Hoechst 33258 (Sigma, Copenhagen, Denmark). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) images were subjected to NIH imageJ software to measure the DNA methylation/DNA content signal by manually outlining the nuclei (n = 2003) of the embryos. The data were analysed using PROC-GLM statistical procedure in SAS 9.1 (SAS Institute Inc., Cary, NC, USA), least square means were compared and P-values were used to decide the significant differences within and between different groups of embryos. The 1-cell stages lacked active demethylation of paternal genome in IV and IVF embryos. Embryos produced under in vitro conditions presented higher levels of DNA methylation than IV. A lineage specific DNA methylation (hypermethylation of inner cell mass and hypomethylation of trophectoderm) observed in porcine IV late blastocysts was absent in PA and SCNT blastocysts despite the occurrence of de novo methylation in early blastocysts. SCNT early (50%) and late (14%) blastocysts presented DNA methylation pattern similar to IV early and late blastocysts, respectively. Concluding, DNA methylation patterns are strongly impaired under in vitro conditions in porcine preimplantation embryos.
40 VARIOUS DNA METHYLATION LEVELS OF IMPRINTED GENES IN CLONED COWS FROM THE SAME DONOR CELLS
Authors: Kaneda M. , Watanabe S. , Akagi S. , Somfai T. , Haraguchi S. , Hirako M. , Geshi M. , Nagai T.
Published: August 2011

Somatic cell nuclear transferred (SCNT) animals are genetically identical to the donors; however, because of epigenetic abnormalities caused by incomplete reprogramming during nuclear transfer, the efficiency of SCNT is still very low. Monozygotic twins are also genetically identical, but it is reported that their epigenetic patterns on the genome, the so-called epigenome, are different. The epigenome is easily influenced by aging, environmental changes and nutrients, therefore these effects can be predicted by comparing epigenetic differences between genetically identical animals. Here we analysed DNA methylation levels of imprinted genes, which express in a parent-of-origin specific manner, in various tissues of cloned cows derived from the same donor cells. Imprinted gene expression is controlled by DNA methylation and other epigenetic modifications and abnormal expression/methylation patterns of imprinted genes have been observed in cloned animals. These alterations also occur during in vitro development of preimplantation embryos, which suggests that imprinted genes are easily influenced by environmental changes. Therefore, we chose H19 and PEG3 imprinted genes for the analysis to determine the epigenetic differences between individual cloned cows derived from the same donor cells. From 5 cloned and 5 non-cloned cows, we isolated DNA from 8 tissues (heart, lung, liver, kidney, spleen, intestine, muscle, and spinal cord) and analysed DNA methylation levels by bisulfite sequencing method. Briefly, genomic DNA was isolated by QIAGEN DNeasy Blood & Tissue Kit and bisulfite converted by QIAGEN EpiTect Bisulfite Kits (Qiagen, Valencia, CA). After amplification, the PCR products were cloned into TA vector and at least 10 clones were sequenced in each gene/sample. In every tissue analysed, the methylation levels largely differ among tissues and individuals. On average, the paternally imprinted gene H19 was 9.4 to 47.9% methylated (average 27.6 ± 10.3%) in clones and 0.5 to 69.8% methylated (average 29.0 ± 16.8%) in non-clones. The maternally imprinted gene PEG3 was 18.8 to 82.2% methylated (average 43.5 ± 15.8%) in clones and 8.0 to 98.7% (average 48.2 ± 18.8%) in non-clones. Even though there were large variations in DNA methylation levels, the variability tends to be low in clones compared to non-clones. More specifically, the variabilities of H19 methylation levels in spleen and intestine were significantly lower in clones than those in non-clones (32.3 ± 5.4% v. 27.0 ± 19.0% and 25.1 ± 4.2% v. 45.1 ± 14.3%, respectively, F-test; P < 0.05). These results suggest for the first time that epigenetic patterns in some tissues of both clones and non-clones are influenced by genetic background; however, mostly they are varied depending on non-genetic factors.
41 DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFERRED RAT EMBRYOS TO THE BLASTOCYST BY A HISTONE DEACETYLASE INHIBITOR, TRICHOSTATIN A
Authors: Kashiwazaki N. , Nakajima N. , Fujimaki K. , Syudo K. , Ito J.
Published: August 2011

Although cloned animals have been reproduced in many species via somatic cell nuclear transfer (SCNT), only 1 previous report showed successful generation of cloned rats. Some researchers evaluated developmental competence of reconstructed rat embryos and showed that these reconstructed embryos were never progressed beyond the 2-cell stage because of inadequate formation of the mitotic assembly and nuclear organisation or improper transcription of cytoskeleton genes. Even though our group also improved the proportion of 2 pronuclear formation in rat reconstructed embryos by the treatment with a proteasome inhibitor, MG132 (Nakajima et al. 2008 Cloning and Stem Cells 10, 461–468), no embryos were developed beyond the 2-cell stage. Recently, it has been demonstrated that a histone deacetylase inhibitor, trichostatin A (TSA) treatment dramatically improves efficiency of cloning in the mouse, although the precise effect of TSA is unclear in SCNT. However, in the case of rats, availability of TSA has not been tested. The aim of the present study was to clarify whether TSA treatment was also effective in rat SCNT. According to our previous reports, rat oocytes were collected and the spindles of the oocytes were removed in medium supplemented with MG132. After enucleation, nuclei of cumulus cells were injected in the oocytes and the reconstructed embryos were cultured in the medium supplemented with 0 (control), 5, 50, or 500 nM TSA. More than 100 embryos are used for each treatment group. Pronuclear formation, development to the 2-cell stage and blastocyst formation were evaluated. Our results demonstrated that TSA treatment affected neither the proportion of pronuclear formation nor development to the 2-cell stage in rat reconstructed embryos. However, the treatment with higher concentrations of TSA treatment (50 nM and 500 nM) compared to the concentration (5 nM) which was usually used for mouse SCNT enabled the reconstructed embryos to develop to the blastocyst stage but at low rate (1.4 to 2.2%). In both the 5 nM treatment and control groups, none of the rat reconstructed embryos developed to blastocyst. Taken together, our data suggests that TSA treatment also seems to be effective in rat SCNT. These findings will be useful for improvement of the protocol in rat SCNT.

This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities to J. I. and N. K.
50 FIRST EQUINE CLONE BORN IN ARGENTINA BY SOMATIC CELL NUCLEAR TRANSFER FROM A POLO ARGENTINO MARE
Authors: Miragaya M. , Revora M. , Rigali F. , Herrera C. , Viviani L. , Quintans C. , Pascualini S. , Losinno L.
Published: August 2011

Births of the first cloned equids were reported in 2003 (Woods et al. 2003 Science 301(5636), 1063; Galli et al. 2003 Nature 424 (6949), 635) but no equine clones have been reported in Latin America. The aim of this work was to inform the birth of the first equine clone in Argentina as a result of interdisciplinary teamwork. Equine oocytes were obtained from slaughterhouse ovaries and were transported at 38°C for 15 h in TCM-199 buffered with 20 mM HEPES and supplemented with 1 mM glutamine, 0.19 mM sodium pyruvate, 2 μg mL–1 LH, 5 μg mL–1 FSH, 100 ng mL–1 EGF, 100 ng mL–1 IGF-I, and 10% FCS. After 16 to 18 h of maturation, oocytes were denuded and enucleated. Frozen–thawed somatic cells from 1 of 2 aged (23 and 20 years old) Polo Argentino breed donor mares were inserted into the perivitelline space of each enucleated oocyte and fusion was induced. Activation was performed by exposure to Ionomycin (5 μM) for 4 min, followed by 4 h culture in a combination of 6-DMAP and cycloheximide in SOFaas culture media (Choi et al. 2001 Reproduction 122, 177–183). Embryos were cultured in 50 μL SOFm droplets for 7 to 8 days and then were transferred non-surgically to anovulatory recipient mares treated with 1.5 mg oestrogen, followed by long acting progesterone (BioRelease P4 300 LA, BET Pharm, KY). The production rates of the first cell line were: fusion 301/553 (54.4%), cleavage 163/218 (53.4%), blastocysts produced 20/163 (12.2%), blastocysts transferred 18/20 (90%), pregnancies 1/18 (5.5%) and no foal born. The production rates of the second cell line were: fusion 252/553 (45.6%), cleavage 55/252 (21.8%), blastocysts produced 8/55 (14.5%), blastocysts transferred 7/8 (87.5%), pregnancies 4/7 (57%) and 1 filly was born. DNA microsatellite analysis confirmed that the filly was a clone from the original donor cell line. The filly, born naturally on Day 329 of gestation, suffered premature placental separation and weighed 12 kg. Despite the intensive care provided, the filly’s respiratory, nervous, and cardiovascular functions deteriorated rapidly and she died 14 h postpartum. Fetal membranes weighed 5.7 kg (47.5% of the filly’s weight) and showed marked oedema of the chorioalantois and umbilical cord (which also showed a torsion of 9 twists). Histopathologically, the chorioalantois showed scant and poorly developed villi and also connective tissue oedema. Death could have been due to marked immaturity, as a consequence of placental insufficiency and umbilical cord torsion.
52 THE EFFECTS OF DONOR CELL CYCLE AND THE TIMING OF OOCYTE ACTIVATION ON DEVELOPMENT OF BOVINE NUCLEAR TRANSFERRED EMBRYOS IN VIVO
Authors: Matsukawa K. , Akagi S. , Fukunari K. , Hosokawa Y. , Yonezawa C. , Watanebe S. , Takahashi S.
Published: August 2011

The cell cycle of donor cells and recipient cytoplasts are important factors affecting development of nuclear transferred (NT) embryos. We previously showed that bovine NT embryos using pre-activated cytoplasts and early G1 cells had a high in vitro developmental rate (SSR, 2008, 41st Annual Meeting). The objective of the present study was to evaluate the effects of donor cell cycle (early G1 or G0 phase) and the timing of oocyte activation on fetal development of bovine NT embryos. Adult fibroblasts from ear skin tissue of Japanese black cattle were used as donor cells. The G0 phase cells were synchronized by serum-starvation, and the G1 phase cells were prepared from actively dividing M phase cells. NT embryo production was performed by 2 kinds of protocols as follows: 1) recipient oocytes were activated by Ca ionophore (CaI), followed with cycloheximide (CH) for 2 h, and fused with synchronized donor cells followed with cytochalasin D (CD) and CH for 1 h, then CH for 4 h (pre-activated), 2) unactivated oocytes were fused with synchronized donor cells and activation was performed by CaI 1 h after fusion, followed by with CD and CH 1 h, then CH for 4 h (post-activated). After activation treatments, NT embryos were cultured in IVD101 medium for 7 days. Then, blastocysts were transferred to recipient cows. Diagnosis of pregnancy was made by ultrasonography at days 30, 60, and 90 (Day 0 = the day of embryo transfer). As shown in Table 1, the blastocyst formation rate of the NT embryos derived from early G1 cells in the pre-activated group was higher than that from G0 cells in the post-activated group (36% v. 23%, P < 0.05). After embryo transfer, 29, 67, and 50% of recipient cows were pregnant at Day 30 in G0 post-, G1 post-, and G1 pre-activated groups, respectively. However, only 1 embryo (14%) of G0 post-activated group developed to term. In conclusion, bovine NT embryos using early G1 cells and pre-activated cytoplasts showed a high blastocyst formation rate, but the full-term development of bovine NT embryos could not be improved by using early G1 cells and pre-activated cytoplasts.

Table 1.Effect of the timing of oocyte activation on developmental ability of bovine NT embryos derived from early G1 or G0 phase cells