bluuebunny/crossref_metadata_2025 huggingface dataset:
298 DIFFERENTIATION OF CANINE AMNIOTIC FLUID MESENCHYMAL STEM CELLS INTO NEURONAL PRECURSORS
Authors: Kim E. Y. , Choi S. A. , Lee J. H. , Kim K. J. , Park K. S. , Park Y. B. , Ha Y. N. , Li X. , Park J. Y. , Kim M. K.
Published: August 2011

The amniotic fluids contain mesenchymal stem cells and can be readily available for tissue engineering. Recently, regenerative treatments such as tissue engineering, cell therapy, and transplantation have shown potential in clinical trials of degenerative diseases. Physiologically, disease presentation and clinical responses in the dog are much more similar to that in the human compared with other traditional mammalian models. In addition, several researchers have demonstrated Canis familiaris is a suitable model for human diseases. The aim of the present study was to investigate whether canine amniotic fluid (cAF)-derived mesenchymal stem cells (MSC) can differentiate into neural precursor cells in vitro by neural induction reagent. The conditions of differentiation of MSC into neural cells were DMEM and N2-supplement, dibutyryl cyclic adenosine monophosphate, and butylated hydroxyanisole. During neural precursor differentiation, cAF-MSC can progressively acquire neuron-like morphology. Expressions of neuron cell-specific markers were examined before and after in vitro induction of differentiation. Changes in mRNA levels of specific genes were quantified by RT-PCR. The mRNA levels of NEFL (730%), GFAP (350%), β-tubuline 3 (2900%), and NSE (960%) were significantly increased after induction. The value of change in mRNA levels before and after induction was evaluated with the Image J program. In addition, the nestin, β-tubuline 3, and tyrosine hydroxylase protein expressions were confirmed by immunocytochemistry assay following the induction of differentiation, compared with the noninduction. In conclusion, this study demonstrated that cAF-MSC have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegeneration diseases including Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.
299 EPIGENETIC REPROGRAMMING OF PORCINE FIBROBLAST CELLS INDUCED BY STURGEON'S OOCYTE EXTRACT
Authors: Kim S. Y. , Park S. H. , Lee M. R. , Eun H. J. , Kim T. S. , Park S. B. , Yoo J. G. , Park C. S. , Lee J. H.
Published: August 2011

The direct conversion of differentiated cells into undifferentiated or pluripotent cells would present scientific and medical benefits because of the potential for customized transplantation therapy. Although somatic cell nuclear transfer is one powerful way to fully reprogram somatic cells into a pluripotent state with the aid of oocyte or egg cytoplasm, the therapeutic applications of this approach have been hindered by technical complications as well as ethical objections. An alternative strategy for epigenetic reprogramming of differentiated cells into pluripotent status is desperately required. We have developed a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte extract to porcine fibroblast cells ex ovo. Porcine fibroblasts were permeabilized by 4 μg mL–1 of digitonin for 2 min at 4°C and then incubated in sturgeon’s oocyte extract for 5 h at 15 to 18°C followed by resealing of the cell membrane. We found that the sturgeon’s oocyte extract induced the reduction of overall levels of tri-methylation at lysine 9 of histone H3 (H3K9Me3), which might be related to preservation of DNA methylation in fully differentiated cells, of porcine fibroblast cells. However, permeabilized porcine fibroblasts after treatment with the extract were increasingly acetylated at lysine 9 on histone 3 (H3K9Ac), which might be associated with expression of pluripotency genes. In addition, the cells treated with the extract showed up-regulation of Oct3/4, Sox2, and Nanog gene expression. When somatic cell nuclear transfer embryos reconstructed by using the treated donor cells were transferred into surrogates, the pregnancy rate was slightly high. These results showed that sturgeon’s oocyte extract can reprogram porcine somatic cells into undifferentiated status. Further work needs to exploit the epigenetic reprogramming of differentiated cells into the undifferentiated state using different species oocyte extract.
306 IN VITRO CARDIOMYOGENIC AND NEUROGENIC DIFFERENTIATION OF MINIPIG BONE MARROW MESENCHYMAL STEM CELLS
Authors: Mohana Kumar B. , Kim T. H. , Lee Y. M. , Maeng G. H. , Jeon B. G. , Ock S. A. , Rho G. J.
Published: August 2011

Differentiation of mesenchymal stem cells (MSC) into specialised cells in vitro before transplantation may improve the engraftment efficiency of the transplanted cells as well as the safety and efficacy of treatment. To understand the differentiation process and the functional identities of cells in an animal model, we examined the in vitro differentiation capacity of porcine MSC (3–6 passage) into cardiomyocyte-like and neuron-like cells. The MSC isolated from the bone marrow of postnatal miniature piglets [T-type, PWG Micro-pig (R), PWG Genetics, Korea] exhibited a typical fibroblast-like morphology and expressed the specific markers, such as CD29, CD44, and CD90. After 21 days of culture in induction media, MSC revealed the appropriate phenotype of osteocytes (von Kossa and Alizarin red), adipocytes (Oil red O), and chondrocytes (Alcian blue). Ther MSC were further induced into cardiomyogenic and neurogenic differentiation following the protocols described earlier (Tomita et al. 2002 J. Thorac. Cardiovasc. Surg. 123, 1132–1140) and (Woodbury et al. 2002 J. Neurosci. Res. 96, 908–917), respectively, with minor modifications. Expression of lineage-specific markers was evaluated by immunocytochemistry, and RT-PCR and quantitative PCR (RT-qPCR). For cardiomyogenic differentiation, MSC were stimulated with 10 μM 5-azacytidine for 24 h, 3 days, or 7 days, and the cells were maintained in culture for 21 days. Upon induction, MSC exhibited elongated and stick-like morphology with extended cytoplasmic processes, and toward the end of culture, cells formed aggregates and myotube-like structures. Immunostaining was positive for the markers of cardiomyocyte-like cells, such as α-smooth muscle actin, cardiac troponin T, desmin, and α-cardiac actin. The RT-PCR and RT-qPCR analysis showed the expression and a time dependent up-regulation of cardiac troponin T, desmin, α-cardiac actin, and β-myosin heavy chain genes. Following induction with neuronal-specific media for 3 days, above 80% of MSC acquired the morphology of neuron-like cells with bi- or multipolar cell processes forming a network-like structure. Induced cells with neuronal phenotype were positively stained for nestin, neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and neurofilament-M (NF-M). The expression of neural transcripts, such as nestin, GFAP, and NF-M, was further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the potential of porcine MSC to differentiate in vitro into cardiomyocyte-like and neuron-like cells, thus offering a useful model for studying their functional and molecular properties before transplantation.

This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.
31 DEVELOPMENT AND APOPTOSIS IN BOVINE CLONED EMBRYOS RECONSTRUCTED WITH OOCYTES COLLECTED BY REPEATED OVUM PICKUP SESSIONS OR FROM SLAUGHTERED COW OVARIES
Authors: Camargo L. S. A. , Pereira M. M. , Quintao C. C. R. , Sales J. N. S. , Iguma L. T. , Serapiao R. V. , Viana J. H. M.
Published: August 2011

The oocyte has important components for nuclear reprogramming and its cytoplasmic background may influence the somatic cell nuclear transfer success. The current study attempted to evaluate the competence of cytoplasm from oocytes recovered by repeated ovum pickup (OPU) in living cows (OPU group) or obtained from ovaries collected at slaughterhouse from unknown source crossbred cows (SH group) to produce nuclear-transferred bovine embryos. For the OPU group, oocytes were recovered from 4 Bos indicus × Bos taurus crossbred cows in 4 repeated OPU sessions. Oocytes of OPU and SH groups were matured in vitro for 17 to 18 h, denuded and exposed to Hoechst 33342 (Sigma, St. Louis, MO, USA) and cytochalasin (Sigma) before enucleation. Embryos of OPU (n = 100) and SH (n = 105) groups were reconstructed with somatic cells from adult Gyr (Bos indicus) cow, fused with double electric pulse of 2.4 kV cm–1 for 30 μs and activated with ionomycin (Sigma) and 6-DMAP (Sigma). Embryos were cultured in CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Cleavage and blastocyst rates were evaluated at 72 h and 168 h post-activation, respectively. Blastocysts at 168 h post-activation were fixed and permeabilized for TUNEL assay (DeadEnd™ Fluorimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. IVF bovine blastocysts (IVF group; n = 245) obtained with oocytes of slaughtered cows were used as control group. Fusion, cleavage, and blastocyst rates were analysed by chi-square test and total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analysed by ANOVA. There were no differences (P > 0.05) in fusion (71.0% and 61.0%), cleavage (74.6% and 78.1%) or blastocyst (32.3% and 31.2%) rates between OPU and SH groups, respectively, but both groups presented greater (P < 0.05) blastocyst rates than the IVF group (15.1%). Total cell number (80.66 ± 5.36 and 82.10 ± 4.79), apoptotic cell number (12.66 ± 3.20 and 15.60 ± 3.04), and apoptotic cell index (0.15 ± 0.03 and 0.20 ± 0.04) were also similar (P > 0.05) between OPU and SH groups, respectively. However, apoptotic cell number (7.40 ± 0.93) and apoptotic cell index (0.07 ± 0.01) were lower (P < 0.05) in the IVF group than the SH group and similar (P > 0.05) to the OPU group. In conclusion, oocytes cytoplasm from both groups (OPU and SH) have the same potential to produce nuclear-transferred bovine embryos but only blastocysts from the OPU group present apoptosis levels similar to its in vitro-fertilized counterpart.

Financial support: Fapemig and CNPq.
316 INDUCTION OF PIG INDUCED PLURIPOTENT STEM CELLS BY RECOMBINANT PROTEINS ENCODED BY DEFINED FACTORS
Authors: Zhang Y. H. , Cao H. G. , Li Y. S. , Yin H. Q. , Sun X. P. , Gui T. , Ji S. F. , Tao Y. , Liu Y. , Zhang X. R.
Published: August 2011

Pluripotent cells derived from any differentiated cell type through ectopic expression of transcription factors were designated as induced pluripotent stem (iPS) cells, exhibiting similar morphology and growth properties to embryonic stem (ES) cells besides expressing ES cell marker. Because iPS have the ability to differentiate into all types of cells, iPS cell technology is thought to have enormous potential for generating disease models, drug screening, toxicology, and regenerative medicine. However, for virus-mediated transfection of defined factors, the exogenous genes generally would be randomly inserted into the target cell’s genome, possibly bringing the potential hazard of insertional mutagenesis. Therefore, it is necessary to seek some new methods to induce somatic cell reprogramming without viruses. With instruction from the work of Zhou et al. (2009 Cell Stem Cell 4, 381–384), in the present study we attempted to use defined factors recombinant proteins-carried cell-penetrating peptide for the generation of porcine iPS cells, which would be a benefit for safe applications of iPS cells. Defined factors genes were amplified by PCR with specific primers of 9 arginines (R9) from recombinant plasmid pLL-hOCT4/pSox2/pMyc/pKlf4-EGFP (Yin et al. 2010 Prog. Biochem. Biophys. 37, 607–612) and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the 4 recombinant plasmids were then transformed into BL21 strains, respectively. After IPTG induction, hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins were purified using Novagen His-Bind kit and confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, respectively. Then, defined factors recombinant proteins were added into pig fetal fibroblasts (PFF) medium every 48 h to establish pig iPS cells. The results showed that purified hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins could enter into PFF efficiently, and most of them were located in nuclei. The PPF were subcultured in stem cell medium condition and treated with defined factors recombinant proteins for 6 cycles simultaneously; the clear-cut cell colonies were gradually derived. These cells had large translucent nuclei and a high nucleo:cytoplasmic ratio and were positive for AP, Oct4, and Nanog. Detailed characterisation of such induced cells is ongoing. This research would provide new ideas for the induction of porcine somatic cell reprogramming.

Y. H. Zhang and H. G. Cao contributed equally. This work was supported by NSFC (30700574 30800784/c120103) and 973 (2009CB941004).
318 EFFICIENCY OF PROTOCOL P-36, ASSOCIATED WITH EQUINE CHORIONIC GONADOTROPIN OR LUTEINIZING HORMONE ADMINISTRATION, IN THE LAST DAY OF SUPERESTIMULATORY TREATMENT, IN NELLORE COWS
Authors: Oliveira A. C. , Mattos M. C. C. , Bastos M. R. , Gonçalves J. R. S. , Lunardi L. H. , Surjus R. S. , Sartori R. , Barros C. M.
Published: August 2011

The objective of this study was to evaluate the use of pLH in replacement of eCG on the last day of P-36 superstimulatory treatment in Nellore donors. Recent studies demonstrated improvement in embryo production when the last 2 doses of FSH were replaced by eCG. However, consecutive use of eCG in bovine superstimulatory protocols may induce antibody against eCG, decreasing embryo production. Twenty-five Nellore cows were randomly allocated in 4 groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent 4 treatments in a cross-over design. Donors received an intravaginal device containing 1.0 g of progesterone (IVD, Primer®, Tecnopec, Sao Paulo, Brazil) and oestradiol benzoate (2.0 mg, IM; Estrogin®, Farmavet, Sao Paulo, Brazil) at a random stage of the oestrous cycle (D0). Cows from the control group were superestimulated with decreasing doses of pFSH (133 mg, IM; Folltropin-V®, Bioniche, Ontario, Canada; D5-8). In the P-36/eCG group, the last 2 doses of pFSH were replaced by 2 doses of eCG (200 IU each dose, IM; Folligon®, Intervet, Boxmeer, the Netherlands). In the P-36/LH2 and P-36/LH4 groups, the last 2 doses of pFSH were replaced by 2 doses of 1 and 2 mg of pLH, respectively (IM; Lutropin®, Bioniche). All animals were treated with prostaglandin F2α (150 μg d-cloprostenol, IM; Prolise®, Tecnopec) on D7, and the IVD was removed 36 h after. Ovulation was induced with 12.5 mg of pLH (IM) on D9, and all animals received fixed-time artificial insemination 12 and 24 h after pLH. Embryo flushing was performed on D16. Data were analysed by ANOVA (Proc Mixed, SAS). There was a significant difference in the number of corpus luteum in the eCG group (19.2 ± 2.4) when compared with the LH2 (12.7 ± 2.0) and LH4 groups (12.3 ± 1.5; P < 0.05). In addition, there was a tendency of lower ovulation rate in the LH2 group as compared with the eCG group (50.6 and 67.8%, respectively; P = 0.06). However, there was no difference in viable embryo yield among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0); P > 0.05. In conclusion, eCG can be replaced by pLH (4.0 mg), in the last day of P-36 protocol, without affecting the production of viable embryos in Nellore cows.

The authors acknowledge FAPESP (Sao Paulo, Brazil) for funding and fellowships for A. C. S. Oliveira, M. C. C. Mattos, and M. R. Bastos.
322 INDUCTION OF SUPEROVULATION IN PROESTRUS DOGS USING SERUM GONADOTROPIN OF PREGNANT MARES AND HUMAN CHORIONIC GONADOTROPIN
Authors: Park K. S. , Kim K. J. , Choi S. A. , Lee J. H. , Kim E. Y. , Park Y. B. , Ha Y. N. , Li X. , Park J. Y. , Kim M. K.
Published: August 2011

Several factors influence the limited application of assisted reproductive technologies (ART) in the canine species. Most problems arise because of the complex nature of reproductive physiology of the dog. For example, dogs are monoestrus, generally exhibiting oestrus only once every 6 month to 1 year. In the canine species, there has been little research on the ART because of difficulties associated with anatomy and reproductive physiology. Because in vitro maturation of canine oocytes has been particularly difficult, in vivo matured oocytes have been used in somatic cell nuclear transfer. However, the number of oocytes that can be obtained using this approach is limited, with ∼6 to 10 good oocytes being obtained per collection. The present study was undertaken to evaluate the effects of different dosages of eCG on folliculogenesis in the dog and to determine the number of oocytes that might be obtained after ovulation. The experimental design involved 3 groups that were treated with different dosages of eCG at the early stage of proestrus; Group A was a nontreatment (control) group, Group B received 200-IU eCG SC injections every day, and Group C was injected with 500 IU of eCG every 2 days until reaching 2 to 3 ng mL–1 serum progesterone concentration, respectively. Dogs in Groups B and C received a 1 000-IU hCG SC injection when progesterone concentrations reached 2 to 3 ng mL–1. The serum progesterone concentration was examined with a Radioimmunoassay Kit (Diagnostic Systems Laboratories Inc., Webster, TX, USA). The day of ovulation was considered as the day when serum progesterone concentration reached 4.0 to 7.2 ng mL–1. Approximately 70 to 76 h after ovulation, the dogs were subjected to the oocyte collection procedure. One-way ANOVA followed by Duncan’s multiple range tests was performed. The significance level was <0.05. In total, 446 oocytes were recovered from 60 bitches, with an average of 6.4 oocytes/dog in Group A (from 49 bitches), 16.6 oocytes/dog in Group B (from 5 bitches), and 8.5 oocytes/dog in Group C (from 6 bitches). The oocytes collection rate (number of oocytes per dog) in Group B was higher (P < 0.05) than those in the other 2 groups. In conclusion, the results showed that eCG treatment with hCG in early-proestrus-stage bitches can result in a greater number of recovered in vivo matured oocytes. This technology could become a useful research tool for canine cloning and ART.
326 CONSTRUCTION OF A SPLICING-DEPENDENT SELECTABLE MARKER FOR GENE TARGETTING
Authors: Cernea S. , Wells K.
Published: August 2011

Gene targeting in mammalian cells plays a crucial role in biotechnology. These experiments are characterised by low rates of homologous recombination and high rates of random integration. Therefore, many fibroblast colonies must be screened to identify a targeting event. To dramatically reduce the survival of random integration events, we have developed a splicing-dependent selectable marker strategy by introducing a mutation in a codon-optimized G418 resistance gene (mNeo). This mutation could be corrected upon homologous recombination. Since the C-terminal region of aminoglycoside phosphotransferase (AphII, Neo/Kan resistance) participates in formation of the active site of this enzyme, we hypothesised that addition of even one amino acid at the C-terminus would render this protein non-functional. To test this hypothesis, a mutation was introduced in an E. coli AphII expression vector that converted the stop codon of AphII to tryptophan (X265W, TGA > TGGTAA). This mutation was confirmed to inactivate AphII by independently characterising the G418 and Kanamycin resistance (or lack thereof) provided by the X265W mutation. To evaluate this mutation in mammalian cells, two intronless mammalian expression vectors were constructed that differed by the presence or absence of the X265W mutation. G418 resistance was only provided by the wildtype sequence, thus confirming that X265W inactivates AphII in mammalian cells. An identical mutation was then introduced into a eukaryotic expression vector based on mNEO. Further, the sequence was extended to create a 5′ intron splice site (TGA > TGGTAAGAGTT). This region was designed to direct splicing between the first and second G residues thus removing the G in the third position of the W codon. The 3′ intron splice sites was then designed to provide an A residue as the first base of the next exon so that successful splicing would correct the mutation by recreating an appropriately positioned stop codon (TGA). To evaluate this strategy in mammalian cells, two plasmids were constructed that harbored the X265W mutation embedded at the 5′ splice site of a downstream intron. In one plasmid (pSC3-G) the first base of the downstream exon begins with a G residue resulting in inactivation of AphII. In the other plasmid (pSC2-A), the first base of the downstream exon begisn with an A residue forming a stop codon that allows for active, wildtype AphII. These plasmids were transfected into porcine fetal fibroblasts and subjected to selection with G418. A positive control plasmid and pSC2-A produced colonies that were too numerous to count. A negative control plasmid and pSC3-G produced no colonies. It can be concluded that the X265W mutation can be corrected by splicing to an exon that begins with an A residue. This splicing-dependent selectable marker may prove useful in gene targeting experiments when the site of modification is followed by an exon that begins with an A.
327 HIGH EFFICIENCY SWINE CLONING USING MONOGENIC AND POLYGENIC POOLS OF GENETICALLY MODIFIED CELLS
Authors: Carlson D. F. , Dobrinsky J. R. , Fahrenkrug S. C.
Published: August 2011

Somatic cell nuclear transfer (SCNT) of genetically modified (GM) cells is currently the most widely applied method for the creation of transgenic swine. However, significant clone-to-clone variation in the efficiency of SCNT for a variety of genetically modified cell lines is commonly observed and contributes to the expense of transgenic animal production. A retrospective look at our own results based on the use of monoclonal GM cell lines as donors revealed a dismal efficiency of only 13% (15 embryo transfers resulting in only 2 full term pregnancies). Thus, while SCNT of individual GM clones offers the perceived advantage of prior characterisation of transgene expression or structure, the variability of clonability for any given cell line adds risk to SCNT. In contrast, rates of pregnancy when we used pools of GM cells as donors for SCNT were much better (9 full term pregnancies from 11 transfers, ∼82% efficiency). Four of these litters were derived from polyclonal but monogenic GM cell populations constitutively expressing either human APOBEC3G or YFP-Cre transgenes integrated using the Sleeping Beauty transposon system. Four litters relied on a novel approach, wherein different polyclonal and monogenic GM cell populations (containing different transgenes) were mixed before being used as donors for SCNT. For example, 2 litters were derived from the pooling of 2 GM cell populations carrying different tetracycline inducible or repressible shRNA transgenes, resulting in founders harboring each of the shRNA transgenes (4 in total). Two litters were also created from a pool of 3 distinct polyclonal cell populations, each harboring a different Cre-lox regulated transgene, resulting in the birth of 11 live piglets with founders corresponding to each of the transgenes. Thus, both mono- and polygenic pooling of GM cells significantly enhances the success of SCNT, largely avoiding variation in cell clonability. Furthermore, pooling results in a significant reduction in the time and number of surrogates required to generate a diversity of genetically modified pigs. Importantly, the use of Sleeping Beauty transgene integration resulted in a high rate of transgene-expressing founders. Where expected, the gene of interest transgenes were expressed in 23 of 29 founders (79%), whereas selectable marker transgene expression was observed in 35 of 40 founders (88%). The combination of efficient SCNT from polyclonal and polygenic cell populations and the high proportion of expressers delivered by Sleeping Beauty transgene integration offer a high-efficiency, low-risk solution to swine transgenesis.
33 PRODUCTION OF CLONED BOER GOATS AND DORPER SHEEP IN ARGENTINA
Authors: Colato C. , Albornoz M. , Mellano M. L. , Mellano P. H. , Mellano J. I. , Meltsas A. , Mellano M. A. , Mellano J. C. , Bordignon V. , Baldassarre H.
Published: August 2011

Somatic cell nuclear transfer (SCNT) has been proposed as an outstanding tool for expanding the dissemination capacity of animals of extreme genetic value, as well as for the genetic resurrection of elite animals affected by incurable disease or that died suddenly. Numbers of outstanding males of meat-specialised breeds of goats (Boer) and sheep (Dorper) recently imported into Argentina were expanded using SCNT technology. Oocytes were collected by laparoscopic ovum pickup (LOPU) from 40 Raza Criolla goats and 38 crossbreed 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% serum, at 38.5°C and 5% CO2. 2 caprine fetal fibroblast cell lines (FF1 and FF2) were established from purebred Boer fetuses generated by selective breeding of elite animals, while a fibroblast ovine line was established from a skin biopsy from an elite Dorper ram. Cells were transferred into previously enucleated oocytes, followed by electric fusion using a single DC pulse of 1.6 kV cm–1 for 70 μs. Finally, the reconstructed embryos were 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, followed by in vitro culture in mSOF media before transfer into the oviducts of synchronized recipients within 24 h after fusion. An average of 10.4 and 12.8 reconstructed embryos were transferred to each of 21 and 12 recipient goats and sheep, respectively. Pregnancy was detected and monitored for the first 3 months by transrectal ultrasound scanning. Initial pregnancy (4 recipients, 33%) was maintained from gestation Day 30 to term in sheep, while goats exhibited a dramatic drop from 9 recipients pregnant (41%) on Day 30 to only 2 (9%) giving birth. Deliveries were by elective C-section. The number of normal offspring with good postpartum survival was 2/2 in goats (100%) and 3/5 (60%) in sheep. Substantial differences were observed between the 2 cell lines used in goats, where pregnancy was 4/11 (36%) for FF1 and 5/10 (50%) for FF2 at Day 30; however, only 2 goats carrying FF2 pregnancies carried. These results are in agreement with previous reports suggesting that cell line may be the largest source of result variation in SCNT. At the time of writing this abstract these clones are ∼4 months of age, healthy and growing normally (>40 kg weight). To the best of our knowledge, these are the first cloned goats produced by SCNT technology in Latin America, and the second group to produce cloned sheep in the region.
331 BLOOD ANTIGEN-COMPATIBILITY BETWEEN CLONED BEAGLES AND TRANSGENIC CLONED BEAGLES
Authors: Kim G. A. , Oh H. J. , Park J. E. , Kim M. J. , Park E. J. , Kim H. J. , Jang G. , Lee B. C.
Published: August 2011

It is known that cloned animals produced by somatic cell nuclear transfer (SCNT) using the same donor cells show immunological compatibility for tissue transplantation. However, immunological compatibility for tissue transplantation or blood transfusion between cloned animals and transgenic cloned animals originating from the same donor cell has not been assessed until now. The objective of this study was to evaluate the compatibility of blood group antigens in cloned dogs with each other, in cloned dogs and transgenic cloned dogs, and in transgenic cloned dogs with each other by a crossmatching test. In Lee’s group, 2 cloned beagles (BG1, 2) were produced from fetal fibroblasts (BF3) using SCNT. Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, although with the transfection of the red fluorescent protein (RFP) gene. All 6 beagles shared the same genetic background except for RFP gene insertion in their genome. Canine blood antigen is labelled as dog erythrocyte antigen (DEA), a critical factor for determining blood antigen compatibility during a crossmatching test. Blood samples were collected from all tested dogs. Serum and red blood cells (RBC) were separated; RBC of all dogs were washed 3 times with 0.9% saline, and a 4% RBC suspension was made from the washed cells. They were mixed in the following experimental designs. In experiment 1, an RBC suspension of cloned beagles was combined with equal volumes of another cloned beagle’s serum. In experiment 2, an RBC suspension of Ruppy 1–3, 5 was mixed with an equal volume of another transgenic cloned beagle’s serum; the reverse reaction was also performed. In experiment 3, an RBC suspension of cloned beagles was mixed with the serum of transgenic cloned beagles and the reverse reaction was performed. All the mixtures were incubated at 37°C for 20 min, centrifuged, and then assessed for hemolysis or agglutination. In experiments 1 and 2, no samples showed any evidence of hemolysis. However, in contrast to the other experiments, experiment 3 showed a different pattern. Although the RBC of transgenic cloned dogs showed hemolysis when mixed with the serum of cloned dogs, the RBC of cloned dogs did not react with the serum of transgenic cloned dogs. From the results, we see that the 4 transgenic cloned beagles could not be donors for blood transfusion, but the 2 cloned dogs could be universal donors for all. In conclusion, cloned beagles and transgenic cloned beagles show blood antigen compatibility within themselves. However, the 4 transgenic cloned beagles showed blood antigen incompatibility with the 2 cloned beagles.

This study was supported by Korean MEST, through NRF (grant #M10625030005-10N250300510), and BK21 program, SNU foundation (Benefactor; RNL BIO) and Natural Balance Korea.
332 INDUCIBLE RED FLUORESCENT PROTEIN (RFP) EXPRESSION IN PORCINE FIBROBLASTS AND TRANSGENIC CLONED EMBRYOS USING piggyBac TRANSPOSITION
Authors: Kim S. J. , Koo O. J. , Park S. J. , Moon J. H. , Kwon D. K. , Kang J. T. , Gomez M. N. , Atikuzzaman M. , Lee B. C. , Jang G.
Published: August 2011

Transgenic pigs are promising animal resources for human disease models and organ donors for xenotransplantation, because they resemble humans anatomically and physiologically. Transgenic pigs have been produced from transfected donor cells using several gene delivery systems including retrovirus infection. Recently, it has been reported that piggyBac (PB) transposition is a highly efficient tool in producing transgenic mice. This study investigated the use of PB transposition to establish transgenic cells and produce transgenic cloned embryos in pigs. We constructed plasmid DNA with red fluorescence protein (RFP) expressed by tetracycline-dependent cassette (from Addgene) with PB site using gateway cloning. We co-transfected porcine fibroblasts with the structured plasmid vector (pB-TET-DsRed), pB-rtTA (from Addgene), and a transposase expression vector pCy43 (Sanger Insitute, Hinxton, UK) using Fugene HD. After 24 h, 2 μg mL–1 doxycycline was added to the culture medium to turn on RFP expression. After 48 h of culture, 1 mg mL–1 neomycin was added to select stable RFP transfectants. Selected fibroblasts were cultured for 9 days without doxycycline, thus reducing RFP expression. After establishment of inducible RFP-expressing cells, the cells were used for somatic cell nuclear transfer. Embryos were cultured in porcine zygote medium-3, and 2 μg mL–1 doxycycline was added 5 days later. As a result, RFP expression was detected in the blastocysts. In conclusion, this study demonstrated that the inducible RFP gene in porcine fibroblasts and embryos was controlled by PB transposition system. Furthermore, this system could be a means of delivering an exogenous gene into porcine somatic cells and embryos for transgenic research.

This study was supported by grants from MKE (#2009-67-10033839, #2009-67-10033805), IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), and BK21 program.
333 OCT-4 EXPRESSION ANALYSIS IN F0 AND F1 PORCINE OG2 TRANSGENICS
Authors: Nowak-Imialek M. , Kues W. A. , Petersen B. , Lucas-Hahn A. , Herrmann D. , Lemme E. , Oropeza M. , Carnwath J. W. , Niemann H.
Published: August 2011

OG2 transgenic pigs provide a new large animal model in which to study Oct-4 expression and the derivation, migration and maintenance of pluripotent cells. They may also prove to be a valuable tool for the development of cell-based therapies. The OG2 transgene consists of the genomic sequence of the murine Oct-4 gene with the enhanced green fluorescence protein (EGFP) reporter gene inserted between the promoter and the coding sequences. As previously reported, 11 OG2 founder animals were produced (7 male and 4 female). Two of the OG2-F0 transgenic boars were mated with 3 wild-type sows and with 2 OG2-F0 transgenic sows. The pregnancy of 1 wild-type sow was terminated at Day 5 after fertilization, and approximately 60% (14/23) of the flushed blastocysts expressed EGFP, demonstrating germ line transmission. The remaining 2 wild-type sows delivered 21 piglets, of which 11 were transgenic. The 2 OG2-F0 sows delivered 9 piglets, all of which were transgenic. Transgenesis and tissue-specific expression of the transgene were determined by Southern blotting, Northern blotting, and real-time PCR analysis. Germ cell-specific expression of the OG2 construct was confirmed in both F0 and F1 transgenics by fluorescence microscopy. Testis isolated from male transgenic piglets exhibited weak EGFP fluorescence in some cells within the seminiferous tubules, whereas testis tissue from adult transgenic boars gave strong EGFP expression in pre-spermatogonial cells. In contrast, fluorescence-activated cell sorting (FACS) analysis and fluorescence microscopy of ejaculated spermatozoa from 3 mature OG2-F0 boars displayed no EGFP fluorescence, as expected. Northern blot analysis of EGFP mRNA revealed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. No EGFP mRNA was detected in other organs or in control testis isolated from wild-type piglets. Real-time PCR and Northern blot analysis showed that the time course and signal intensity of EGFP expression in OG2 testis paralleled expression of the endogenous Oct-4 gene in both transgenic and in wild-type testis, confirming that there is indeed stronger expression of Oct-4 in the adult testis than in testis from younger animals. We conclude that the OG2 founders exhibit germline transmission and that the offspring express EGFP in a pattern that faithfully mimics expression of the endogenous Oct-4 gene, thus providing a marker for pluripotent cells. We are currently using FACS to isolate EGFP-positive germ cells (pre-spermatogonial stem cells) from the testis of OG2 boars for further characterisation.

Funded by BMBF.
336 TRANSGENIC Stra8-EYFP PIGS: A MODEL FOR DEVELOPING MALE GERM CELL TECHNOLOGIES
Authors: Sommer J. R. , Jackson L. , Simpson S. , Collins E. B. , Piedrahita J. , Petters R. M.
Published: August 2011

Stimulated by retinoic acid 8 (STRA8) is a protein that is required for meiotic initiation in both male and female gametes in vertebrates. It is also expressed in embryonic germ cells and neonatal male germ cells of mice. The utility of using the Stra8 promoter to recognise and isolate pre-meiotic male germ cells has been reported by others in the mouse. In order to mark germ cells in male pigs, we cloned 1.6 kb of the mouse Stra8 promoter and used it to develop a reporter plasmid using mitochondrial-localised enhanced yellow fluorescent protein (mEYFP). The Stra8-mEYFP transgenic male pigs were produced using somatic cell nuclear transfer. The mEYFP reporter was expressed and easily detectable in the live germ cells of the mature animals and could be observed during tissue culture. The mitochondrial-localised expression of the EYFP reporter was helpful in observing the size and stage of the germ cell. The mEYPF protein was found to be expressed only in the testis of the transgenic pigs using Western blot analysis, whereas endogenous STRA8 protein was also detected in the lung and brain. Fluorescent immunohistochemistry of testicular sections of the transgenic pigs indicated a similar expression pattern to that of the endogenous STRA8 protein. There was an overlap in the expression of the mEYFP and the endogenous STRA8 protein; however, it was observed that the mEYFP protein was present at an earlier stage of spermatogenesis than the STRA8 protein. Immunocytochemistry performed on plated tubules similarly showed varying intensity in expression between the mEYFP transgene and the endogenous STRA8. The difference in the timing of protein expression may be due to the model created or the use of the mouse Stra8 promoter for the expression of mEYFP. Alternatively, the lag in expression between that of the endogenous STRA8 and mEYFP protein may be due to attenuated translation of the Stra8 mRNA. This transgenic model should be useful for the study of reproduction, development, transplantation, biotechnology, and culture of the pig male germ line.

Supported by North Carolina Agricultural Research Service 02234.
339 BIOENGINEERING OF THE MAMMARY GLAND OF LIVESTOCK: INCREASED PROPEPTIDE PROCESSING OF FACTOR IX IN THE MILK OF TRANSGENIC PIGS BY CO-EXPRESSION OF FURIN
Authors: Zhao J. , Walters E. , Calcaterra J. , Ross J. , Spate L. , Samuel M. , Rieke A. , Murphy C. , Butler S. , Velander W. , Prather R.
Published: August 2011

Coagulation Factor IX is a vital protein that plays an important role in the blood coagulation cascade. Hereditary deficiency in the Factor IX gene can result in hemophilia type B, the second most common hemophilia. The milk of transgenic livestock can be an efficient vehicle for producing complex, post-translationally modified blood proteins. The advantages include decreased pathogen risk as well as 100-fold or more production efficiency over blood fractionation and cell culture biotechnology. Although the mammary gland can make many of the complex post-translational modifications necessary for biological function of blood proteins, improvements in propeptide cleavage and decreased proteolytic degradation are desirable. To explore the possibility of producing bioactive human Factor IX protein in pig milk, male and female Landrace fetal fibroblast cells were co-transfected by electroporation with 3 different transgene constructs, Factor IX (FIX), Furin, and SERPINA1. The SERPINA1 construct (containing a Neo selectable marker) was delivered into fetal fibroblasts at a 10-fold-lower molar concentration than the other 2 constructs. Following selection in Geneticin, the presence of all 3 genes was verified by PCR and then cells were used as donors for somatic cell nuclear transfer. Thirteen F0 female piglets from 3 potential different integrations were delivered, and 6 piglets were validated by PCR to be positive for all 3 genes. Among the 6 transgenic pigs, 3 are healthy and able to reach puberty. Milk was collected by induced lactation from 2 gilts. A short murine whey acidic protein promoter-Furin gene was used to limit Furin to the lowest levels needed for pro-FIX processing. Furin was expressed to increase propeptide cleavage efficiency, with the result being complete processing of pro-FIX to FIX at ∼0.3 g L–1 pro-FIX. Total FIX levels were ∼1 g L–1. SERPINA1 was also co-expressed at ∼1 g L–1 or more and this serine protease inhibitor did not seem to inhibit furin processing of the pro-FIX. Fifteen F0 male piglets from 3 potential different integration sites were delivered and all of them were positive for all 3 genes. Four F0 males were chosen to breed with wild-type females, and 5 litters of F1 piglets were born. Of 63 F1 piglets, 22 were tri-transgenic and 3 were di-transgenic (only carrying FIX and Furin). Two F1 females were mated with wild-type males, are confirmed to be pregnant, and will be used to determine the expression level and bioactivity of the Factor IX protein in the milk.

Funded by the NIH NCRR (RR018877) and R01 HL078944.
37 LIMITATION OF BOVINE OOPLASM IN REPROGRAMMING OF PORCINE SOMATIC CELLS
Authors: Østrup O. , Strejcek F. , Petrovicova I. , Morovic M. , Laurincikova N. , Lucas-Hahn A. , Petersen B. , Niemann H. , Laurincik J. , Hyttel P.
Published: August 2011

Ooplasm posses several, undefined factors allowing for reprogramming of somatic genome into pluripotent state. However, it is presently unknown if and to what extent the ooplasm is able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). The 1-cell stage embryos were processed at different time points (2, 4, 8, and 12 h post-activation) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualisation of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). The embryos produced by SCNT of porcine fibroblast into non-activated bovine ooplasm were compared to bovine parthenogenetic counterparts. In the absence of morphological remodelling features (premature chromatin condensation, nuclear envelope break-down), intergeneric embryos showed bovine nuclear and nucleolar precursor body (NPB) morphology. The somatic cell introduced into the bovine ooplasm displayed intranuclear activity at the post-translational level signalling a universal function of ooplasmic factors. The lack of distinct UBF localization in intergeneric embryos, however, suggests failures in sequence-specific interactions between the ooplasm and transferred cell from another genus. We conclude that sperm or a somatic cell of the same species is required for successful embryonic development.

Clonet MRTN-CT-2006-035468; MedRat LSHG-CT-2006-518240; PluriSys FP7-HEALTH-2007-223485; DFG, Alexander von Humboldt fellowship, VEGA 1/0057/08; VEGA 1/0012/10.
39 IN VITRO DEVELOPMENT OF CANINE EMBRYOS PRODUCED BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER USING ENUCLEATED BOVINE OOCYTES
Authors: Kaedei Y. , Fujiwara A. , Tanihara F. , Namula Z. , Vien V. L. , Otoi T.
Published: August 2011

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleous-cytoplasm interactions, and may provide an alternative for cloning endangered animals, whose oocytes are difficult to obtain. Using readily available oocytes from domestic/farm animals as recipients for iSCNT would greatly benefit ongoing research on somatic cell reprogramming. However, little information is available concerning the development of canine iSCNT embryos reconstructed with bovine oocyte cytoplasm. In the first experiment, we investigated the influence of donor cell type on the development of canine iSCNT embryos reconstructed with enucleated bovine oocytes. Canine mammary gland tumour (MGT) cells and cumulus cells were used as donor cell. The bovine oocytes matured for 22 h were enucleated by the micromanipulator, and the donor cells were transferred into the perivitelline space adjacent to the plasma membrane of the oocyte. The couples were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 μs, using an electro cell fusion generator. The reconstructed embryos were cultured for 72 h in the mSOF medium supplemented with 0.4% BSA. After 72 h of culture, only cleaved embryos were further co-cultured with bovine cumulus cells in mSOF supplemented with 5% fetal bovine serum (FBS) for an additional 5 days. In the second experiment, we examined the effects of serum type on the development of canine iSCNT embryos. The embryos reconstructed with canine cumulus cells were co-cultured with canine cumulus cells in mSOF supplemented with 5% FBS, and canine oestrous and diestrous serum for 5 days after 72 h of culture with 0.4% BSA. Data were analysed by chi-square analysis with a Yates’ correction. More than 75% of the canine somatic cells successfully were fused with bovine enucleated oocytes following electrofusion, irrespective of the types of the donor cells. There were no significant differences in the cleavage rates of iSCNT embryos between the cumulus cell and MGT cell (66.2% v. 62.6%). Although none of the embryos reconstructed with MGT cells (n = 123) developed to the 16-cell stage, 6% of embryos with cumulus cells (n = 133) reached at least the 16-cell stage. There were no significant differences in the cleavage rates of iSCNT embryos among the types of serum. The iSCNT embryos could not develop to the blastocyst stage, irrespective of the type of donor cell and serum. In conclusion, our results indicate that the bovine oocytes partly supported the remodelling and reprogramming of the canine somatic cell nuclei, but they were unable to support the development to the blastocyst stage of canine iSCNT embryos. Moreover, the development to the late embryonic stage of iSCNT embryos may be influenced by the type of donor cell but not serum.