bluuebunny/crossref_metadata_2025 huggingface dataset:
232 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE ON IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Maziero R. R. D. , Guastali M. D. , Sudano M. J. , Paschoal D. M. , Guasti P. N. , Crocomo L. F. , Papa P. M. , Listoni A. J. , Papa F. O. , Landim Alvarenga F. C.
Published: December 2012

Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes without having a negative effect on subsequent development to the blastocyst stage. The aim of this study was to evaluate the in vitro production of bovine embryos in the presence of BL-I (Enzo Life Sciences International Inc., Plymouth Meeting, PA, USA) and ROS (Sigma-Aldrich, St. Louis, MO, USA). For that, Nellore oocytes were matured in TCM-199 with Earle’s salts + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in the presence of 25 µM BL-I + 6.25 µM ROS. The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand), in a 5% CO2 atmosphere. Semen was selected through Percoll gradient and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured inside a bag in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocyst rate was evaluated. Five replicates were done. Data were analysed with ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate: control, 33.3 ± 3.74%; BL-I + ROS, 38.0 ± 4.5%. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes did not compromise their subsequent developmental competence. The next steps of the present study are evaluating the kinetics of blastocyst formation and detecting apoptotic cells in situ by TUNEL, which will show that these substances do not compromise embryonic quality.
238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS
Authors: Rho N. Y. , Ashkar F. A. , Revay T. , Madan P. , King W. A.
Published: December 2012

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated.
Funding was provided by CRC, NSERC, and the EmbryoGENE network.
273 BOVINE EMBRYO SEXING IN FIELD CONDITIONS: EFFICACY OF THE POLYMERASE CHAIN REACTION METHOD AND PREGNANCY RATES IN DAIRY HERDS LOCATED IN THE SOUTH AND SOUTHEAST REGIONS OF BRAZIL
Authors: Alvarez R. H. , Cardoso C. R. S. , Butzke G. , Sousa R. V.
Published: December 2012

Currently, artificial insemination with sexed semen is the simplest and most economical strategy for choosing the sex of the offspring. However, sexed semen is not available for certain valuable bulls. Therefore, the use of unsexed semen and subsequent sexing of embryos before transfer into recipients can be an interesting approach to take advantage of these bulls. This study aimed to evaluate the efficiency of embryo sexing under field conditions, as well as the technical performance of dairy farms in the south and southeast regions of Brazil. Day 7 embryos (n = 2447) collected from superovulated (porcine FSH-prostaglandin F2α protocol) cows kept in 8 herds in the states of Paraná and São Paulo were morphologically classified (grades 1, 2, and 3) and subjected to micromanipulation to remove a biopsy of trophoblast cells. Biopsied embryos were kept on standby in holding medium (TQC, Nutricell, Campinas, São Paulo, Brazil) until sexing results were known. Each biopsy was introduced into individual plastic microtubes containing 8 mL of lysis solution and proteinase K (Invitrogen, Darmstadt, Germany) maintained at 55°C for 5 min, followed by 5 min at 95°C. After this step was added 20 µL of a solution composed of primers specific to the Y chromosome (5′-CCTCCCCTTGTTCAAACGCCCGGAATCATT-3′; 5′-TGCTTGACTGCAGGGACCGAGAGGTTTGGG-3′) and for an autosomal gene (5′-AAGACCTCGAGAGACCCTCTTCAACACGT-3′; 5′-AGGTCGCGAGATTGGTCGCTAGGTCATGCA-3′), deoxynucleotide 5′-triphosphates, reaction buffer, and a Taq DNA polymerase kit (Embrapa, Brasília, Brazil). The samples were transferred to a thermal cycler and subjected to 40 cycles of temperature variation for DNA amplification. After PCR amplification, 20 µL of each sample was applied in 2% agarose gel and subjected to electrophoresis (100 V, 40 mA) for approximately 35 min. After identifying the sex, the biopsied embryos as well as additional intact embryos (n = 3945) were transferred into recipients programmed to be on Day 7 of oestrous. The diagnosis of pregnancy and confirmation of the sex of fetuses were performed by ultrasonography between 60 and 70 days after embryo transfer. Pregnancy rate differences after the transfer of intact and biopsied embryos were analysed by a chi-squared test. The reading of the electrophoresis band for sex identification was clear for 2202 (90%) embryos and doubtful for 245 embryos. The overall pregnancy rates were 55.9 and 58.3% for biopsied and intact embryos, respectively (P > 0.05). Pregnancy rate from sexed Grade 3 embryos (32.5%, 67/206) was lower (P < 0.05) than those from Grade 1 (60.1%, 834/1388) and Grade 2 (53.3%, 463/853) embryos. The accuracy of sex identification (sex predicted by PCR and sex observed by ultrasonography) was 93.2%. These results indicate that embryo sexing by PCR can be used successfully in on-farm conditions.
274 USE OF DOPPLER ULTRASOUND FOR EQUINE FETAL SEX DETERMINATION
Authors: Resende H. L. , Carmo M. T. , Alvarenga M. A.
Published: December 2012

Fetal sex determination in horses is increasingly practiced because of commercial interests, such as obtaining offspring of a desired sex. However, in the last several years, the sex determination technique has been done exclusively by genital tubercle evaluation (55 to 70 days of pregnancy), and it has begun to be done more frequently in horses through evaluation of the fetal gonad (100 to 160 days of pregnancy) because of the difficulties of finding the genital tubercle and the large size of the fetal gonad around 4 months of pregnancy. Doppler ultrasound constitutes a practical, effective, and non-invasive technique for real-time blood flow evaluation of the reproductive tract in horses. The aim of this work was to compare two methods of ultrasound to diagnose equine fetal sex between 100 and 140 days and between 140 and 160 days. Evaluations were performed in 112 pregnant mares between 100 and 160 days of pregnancy using B-mode and colour Doppler ultrasound (My Laboratory Five®, Esaote, Nutricell, Campinas, São Paulo) transrectally. The accuracy of sex identification by ultrasound was successfully tested after birth in all the fetuses analysed (44 females and 68 males). By B-mode ultrasound, it was possible to visualise the gonad in all fetuses (44/44) of females between 100 and 160 days of pregnancy. In males, during the period of 100 to 140 days of pregnancy, gonads and mediastinum were observed in all male fetuses (48/48) of evaluations, and between 141 and 160 days of pregnancy, gonads were detected in 100% (20/20) of fetuses and the mediastinum was detected in 30% (6/20). With colour Doppler ultrasound, the vascularization of the female gonad (between the cortical and medullary zones) was observed in 100% (44/44) of females. In males, between 100 and 120 days of pregnancy, the vascularization of the pampiniform plexus and testicular vein were observed in 100% (28/28) of cases. During the period between 121 and 140 days, vascularization of the pampiniform plexus was observed in 100% (20/20) of the fetuses, and vascularization of the testicular vein was observed in 90% (18/20). When evaluated between 141 and 160 days of pregnancy, vascularization of the pampiniform plexus was detected in 100% (20/29) of male fetuses and vascularization of the testicular vein was detected in 80% (8/10). There was no statistical difference between results obtained with B-mode and colour Doppler ultrasound. Both techniques had high accuracy for equine fetal sex determination. However, according to information from the operators, the colour Doppler ultrasound allowed for a faster and more practical exam because visualisation of tissue blood perfusion facilitated the anatomical assessment of fetal structures. In summary, colour Doppler ultrasound is an effective technique for equine fetal sex determination between 100 and 160 days of pregnancy.
28 EFFECT OF TRICHOSTATIN A ON IN VITRO EMBRYO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED FROM CAT DONOR NUCLEI AND BOVINE CYTOPLASM
Authors: Wittayarat M. , Namula Z. , Luu V. V. , Do L. T. K. , Sato Y. , Taniguchi M. , Otoi T.
Published: December 2012

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus-cytoplasm interactions and may provide an alternative for cloning endangered animals whose oocytes are difficult to obtain. The developmental ability of iSCNT embryos decreases with increases in taxonomic distance between the donor and recipient species. The development of cat-bovine iSCNT embryos is reportedly blocked at the 8-cell stage (Thongphakdee et al. 2008 J. Reprod. Dev. 54, 142–147). Abnormal epigenetic reprogramming, such as DNA methylation or histone modifications, may cause low iSCNT efficiencies. The present study was conducted to evaluate the effect of the histone deacetylase inhibitor trichostatin A (TSA), previously used to enhance nuclear reprogramming following SCNT, on the developmental ability of cat iSCNT embryos using bovine oocytes matured in vitro. The matured bovine oocyte was enucleated by the glass needle and the domestic cat fetal fibroblast used as the donor nuclei was then placed into the perivitelline space adjacent to the plasma membrane of the oocyte. Couplets with bovine ooplasm were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 µs, respectively, using an electro cell fusion generator followed by cycloheximide treatment. Reconstructed cat-bovine embryos were treated with 0, 25, 50, and 100 nM concentrations of TSA for 24 h following fusion. The percentages of embryos cleaved and embryos developed to the blastocyst stage were subjected to arc sin transformation before ANOVA. The TSA treatment at 50 nM contributed significantly higher rates of cleavage and blastocyst formation (n = 139; 84.3 and 4.6%, respectively) compared with untreated embryos (n = 187; 63.8 and 0%, respectively) and embryos treated with 100 nM TSA (n = 172; 71.4 and 0%, respectively; P < 0.05). Development to the morula stage of iSCNT embryos was observed in the TSA treatment groups, whereas no embryos developed beyond the 16-cell stage in the untreated group. In conclusion, our results indicate that TSA treatment for 24 h following fusion improves the development of iSCNT embryos. Specifically, 50 nM TSA treatment provides a beneficial effect on cleavage and development to the blastocyst stage of cat iSCNT embryos using bovine oocytes matured in vitro as recipients and domestic cat fibroblasts as donor nuclei.
281 USE OF SMALL MOLECULES ENHANCES REPROGRAMMING SUCCESS IN BOVINE DERMAL FIBROBLASTS
Authors: McMillan M. E. , Grace A. , Andronicos N. , Hinch G. , Schmoelzl S.
Published: December 2012

Reprogramming of differentiated cells to induced pluripotency holds tremendous potential for livestock reproduction. Here we report use of small molecules to support the generation of stem cell-like colonies from bovine dermal fibroblasts that show characteristics of pluripotent cells. Reprogramming of bovine somatic cells was undertaken by introducing canonical reprogramming factors Oct4, Sox2, Klf4, and c-Myc through a lentiviral drug-inducible polycistronic vector. Several small molecules were tested to support reprogramming. A combination of 3 small molecules (sodium butyrate, PD0325901, and SB431542; NaB-PD-SB) in iPS medium [Minimum Essential Medium Alpha, 20% fetal bovine serum, 1× insulin-transferrin-selenium (ITS), 2 mM Glutamax, 100 µM NEAA, 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the kinetics of the reprogramming process. Colonies appeared at 12 days of culture compared with 21 days in iPS medium alone (P < 0.01, n = 5). Colonies in NaB-PD-SB iPS medium had consistent morphology, with small cells in compact dome formations with well-defined colony borders. Cells were not passaged but were either harvested for mRNA extraction or differentiated into embryoid bodies. Gene expression of the reprogramming factors in stem cell-like colonies was confirmed by qRT-PCR, and further gene expression analysis revealed activation of Nanog and ALPL mRNA expression (P < 0.01, n = 2). Embryoid bodies were analysed for gene expression by quantitative RT-PCR. Silencing of the transgene was not observed in embryoid bodies despite withdrawal of the inducer doxycycline. Expression of the endoderm marker FOXA2, ectoderm markers NES and TUBB3, and mesoderm marker DES was strongly increased in embryoid bodies compared with non-reprogrammed bovine fibroblasts (P < 0.01, n = 2), confirming expression of marker genes for the 3 germ layers.
29 EFFECT OF EMBRYO CULTURE LENGTH ON PRODUCTION OF CLONED TRANSGENIC GOATS
Authors: Hall J. , Meng Q. , Sessions B. R. , Fan Z. , Wang X. , Stott R. , Rutigliano H. , Davies C. J. , Panter K. , Bunch T. , White K. L. , Polejaeva I. A.
Published: December 2012

The yield of blastocysts and hatched blastocysts using in vitro production (IVP) in goats are still low. The development of caprine embryos is frequently arrested at the 8- to 16-cell stage, indicating suboptimal culture conditions (Jimenez-Macedo et al. 2005 Theriogenology 64, 1249–1262). Our goal was to produce transgenic goats by somatic cell nuclear transfer (SCNT) and further determine whether the length of embryo culture has an effect on development to term. We compared the efficiency of transferring single-cell embryos 12 h post-activation to transferring 4- to 8-cell embryos cultured for 60 h post-activation. Nine transgenic goats from 2 cell lines were produced through SCNT. Somatic donor cells were obtained from 2 sources: adult fibroblasts and fetal fibroblasts. Adult fibroblasts were obtained from a transgenic doe skin biopsy. Fetal fibroblasts were isolated from a 25-day-old fetus and then electroporated with a pcDNA3.1DV5-MHC-TGF-β1cys33ser vector, followed by G418 selection, screening, and subsequent use for SCNT. Oocytes with >4 layers of cumulus cells were collected by slicing abattoir ovaries and matured in vitro for 21 to 23 h. After being denuded, oocytes presenting a first polar body were enucleated and received a donor cell from 1 of the 2 cell lines. Fused embryos were then activated for 5 min in 5 µM ionomycin, followed by 4 h in 2 mM DMAP with 5 µg of cycloheximide mL–1. Activated embryos were cultured in G1 medium with 5 mg of BSA mL–1 for either 12 or 60 h post-activation, followed by surgical transfer into the oviducts of recipients synchronized to show estrus within 12 h of SCNT. Overall, 376 embryos were transferred into 23 recipients. Pregnancy was examined by ultrasonography on Day 30 post-transfer. No pregnancy losses were observed after Day 30 of gestation. All kids were born live (42% of recipients receiving embryos cultured for 12 h gave birth, compared with only 9% when cultured for 60 h). The data (Table 1) suggest that a longer culture time in vitro significantly reduces viability of cloned embryos.

Table 1.Twelve-hour versus 60-h embryo culture

This work was supported by Utah Agricultural Experiment Station project no. 1100.
299 A FEATURE OF SELF-RENEWAL PORCINE EMBRYONIC STEM CELL-LIKE CELL LINES ESTABLISHED BY INHIBITORS
Authors: Haraguchi S. , Tokunaga T. , Furusawa T. , Ohkoshi K. , Nakai M. , Ikeda M. , Kikuchi K. , Dang-Nguyen T. Q. , Somfai T. , Akagi S. , Kaneda M. , Hirao Y. , Watanabe S. , Geshi M. , Nagai T.
Published: December 2012

Despite meticulous attempts for more than two decades, establishment of authentic porcine embryonic stem cell (ESC) from pig has never been successful. Although putative porcine ESC-like cells have been reported, such cell lines easily lose the ability of self-renewal, becoming extinct or differentiating after only a limited number of passages in culture. Porcine ESC-like cells exhibiting the property of self-renewal rather than pluripotency are considered a valuable resource in applications such as drug screening and toxicology testing in humans and livestock, and in veterinary medicine. In the present study, we evaluated the effect of glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 and Erk signalling inhibitor PD184352 for use in establishing ESC-like cell lines derived from the inner cell mass (ICM) of porcine blastocysts produced in vitro. These ICM-derived cell lines were initially cultured and passaged in conventional human ES medium. They displayed so-called ESC-like morphology; for example, the isolated colonies began to grow as a monolayer with coarse cell–cell boundaries, in which the cells exhibited polygonal boundaries, high nuclear/cytoplasmic ratios, abundant lipid-like inclusions, alkaline phosphatase activity, and expression of markers of undifferentiated cells such as OCT4 and NANOG. After transfer to culture in ES medium containing the inhibitors, the morphology of the colony was dramatically changed, displaying a closely packed and smooth-edged colony with tight cell–cell boundaries. Remarkably, growth of the established cell lines is leukemia inhibitory factor (LIF)-dependent. The inclusion of inhibitors supports self-renewal, thus enabling continuous culture for over 100 passages while maintaining an undifferentiated state. High-passage-number cells continued to express undifferentiated marker genes and showed alkaline phosphatase activity and telomerase activity with an X chromosome status of XaXi. We further investigated the potential for differentiation of the established cell lines. The cells could easily form embryoid body-like spheres in suspension culture. When either the spheres or ESC-like cells were inoculated under the kidney or testis capsules of nude mice, classical teratoma formation was not observed after 2 to 3 months. However, histological analyses revealed apparent invasive proliferation derived from porcine cells. Although further analyses are required to characterise the property of the porcine ESC-like cells, we have recently succeeded in establishment of green fluorescent protein (GFP)-expressing stable cells lines, which will be useful for further investigation.
301 THE VOMERONASAL ORGAN AS A NEW SOURCE OF STEM CELLS FOR CELL THERAPY
Authors: Rodrigues M. N. , Pignatari G. C. , Grondona K. E. B. , Carvalho R. C. , Miglino M. A.
Published: December 2012

The vomeronasal organ (VNO), part of the olfactory system, is located at the base of the nasal cavity at both sides of the nasal septum. The VNO has a tubular shape and possesses receptors to detect pheromones that enable animals to modulate social and reproductive behaviour. Its sensory cells show neurogenesis throughout life, supplied by proliferating stem cells situated at the basal layer that migrate to the surface and generate sensory neurons. The aim of this study was to isolate and characterise stem cells from the VNO in a laboratory animal model species, the New Zealand rabbit. Five males were used for cell isolation and cultivation of stem cells. Three culture media were tested: α-MEM, DMEM-F12, and DMEM-high glucose. As typical for stem cells, isolated cells had a fibroblastic shape, were adhered to the plastic, and achieved good convergence. The cells expanded in all 3 culture media at Day 13. At Day 20, they reached sufficient confluency to promote cell growth and trypsinization. Microscopic analysis revealed that confluence and cell growth were best in DMEM-high glucose medium, confirmed by the proliferation assay (MTT). Cultured stem cells differentiated functionally into adipocytes, osteocytes, and chondrocytes. They did not form tumours when injected into immunocompromised nude mice. In flow cytometry, the cultured cells were negative for CD45 and CD34 and positive for CD105, CD90, Oct3/4, and Stro-1, characterising them as mesenchymal cells. In immunocytochemistry, positive staining was observed for Nanog, vimentin, cytokeratin, Oct3/4, and GFAP. In conclusion, the expression of mesenchymal stem cell markers, the nonexpression of endothelial and haematopoietic markers, as well as the classical patterns of differentiation (adipo-, osteo-, and chondrocytes) and growing processes, indicated that these cells were mesenchymal stem cells. Thus, they may have high therapeutic potential for cell therapy in animals with problems related to reproduction and olfactory function.
312 INFLUENCE OF LARGE NONOVULATED FOLLICLES ON ESTROUS INDUCTION AFTER EMBRYO COLLECTION IN SUPEROVULATED JAPANESE BLACK COWS
Authors: Taniguchi M. , Sasaki E. , Otoi T.
Published: December 2012

Rapid oestrous induction after embryo collection shortens the calving interval of donor cows and improves the efficiency of embryo production. This study aimed to evaluate whether large nonovulated follicles present at the time of embryo collection affect oestrous induction after embryo collection in Japanese Black donor cows. Superovulation treatments were initiated during mid-cycle (9–12 days after oestrus) with 20 AU of FSH (Antrin; Kyoritsu Seiyaku Corp., Tokyo, Japan) administered intramuscularly through 6 injections of decreasing doses at 12-h intervals. On the third day, the cows received 0.5 mg of prostaglandin F2α (PGF; Resipron-C, Asuka Seiyaku Corp., Tokyo, Japan) to induce regression of the corpus luteum and were artificially inseminated 60 to 72 h later. Embryos were collected 7 days after AI. All cows received 0.5 mg of PGF immediately after embryo collection and were allocated to 1 of 2 groups: cows (n = 6) with one or more large nonovulated follicles >18 mm in diameter (Group 1) and cows (n = 5) with no large follicles (Group 2). Oestrus detection was performed by visual observation conducted twice daily after embryo collection. Ovarian ultrasonographic examinations and blood collections (for serum progesterone [P4] and oestradiol [E2] measurements) were performed once daily until the next ovulation. Statistical significance was determined using Student’s t-test. There were no differences in the numbers of corpora lutea, ova/embryos, and transferable embryos or the percentages of transferable embryos between the 2 groups. The interval (mean ± SEM) from embryo collection to the appearance of the dominant follicle and to ovulation was longer (P < 0.05) in Group 1 (5.3 ± 0.7 and 11.0 ± 1.0 days, respectively) than in Group 2 (2.6 ± 0.7 and 8.2 ± 0.7 days, respectively). However, the interval from embryo collection to luteolysis (P4 <1 ng mL–1) did not differ between the 2 groups (4.0 ± 0.7 v. 3.6 ± 0.7 days). In conclusion, the presence of large nonovulated follicles at the time of embryo collection may delay oestrous induction after embryo collection.
326 GENERATION OF A DOUBLE-TRANSGENIC PIG WITH PANCREAS-SPECIFIC GREEN AND LIVER-SPECIFIC RED FLUORESCENCE
Authors: Matsunari H. , Nakano K. , Kanai T. , Sakai R. , Watanabe M. , Umeyama K. , Kobayashi T. , Yamaguchi T. , Shiota A. , Nagaya M. , Nakauchi H. , Nagashima H.
Published: December 2012

Transgenic (Tg) pigs with organ/tissue-specific fluorescence expression provide invaluable research tools for many types of studies, such as organogenesis analysis, in vitro tissue generation from pluripotent cells, and progenitor/stem cell transplantation therapy. We aimed to develop a Tg pig characterised by pancreas- and liver-specific fluorescence expression. A 8.4 kb transgene construct expressing Venus (green fluorescence) under the control of the mouse Pdx1 (pancreatic duodenal homeobox-1) promoter and a BAC-derived construct (170 kb) consisting of the whole-length porcine albumin (Alb) promoter and humanized Kusabira-Orange (huKO, red fluorescence) was introduced into porcine in vitro-matured oocytes using the intracytoplasmic sperm injection (ICSI)-mediated gene transfer method. Injected embryos were transferred to the oviducts of oestrus-synchronized recipients after culture for 1 to 3 days. The transfer of 370 Pdx1-Venus embryos into 4 recipients produce 22 (5.9%) fetuses/piglets, and 9 (40.9%) Tg pigs exhibited pancreas-specific Venus expression. Two (1 male and 1 female) founder Pdx1-Venus-Tg pigs were mated with wild-type (WT) pigs and produced 32 offspring in 3 litters, of which 16 (50.0%) were transgenic. Pancreas-specific Venus expression was inherited in these Tg offspring. The transfer of 523 Alb-huKO embryos into 4 recipients resulted in 19 (3.6%) piglets including a Tg female, which showed liver-specific huKO fluorescent expression. Expression of huKO was detected by RT-PCR exclusively in liver, but not in 7 other organs/tissues examined, including heart, lung, stomach, small intestine, spleen, kidney and skin. This founder Tg female produced a total of 12 non-Tg and 5 Tg offspring (in 2 litters) after mating with a WT boar. Liver-specific huKO expression was inherited in these Tg offspring. Furthermore, the mating of a female Pdx1-Venus pig with an Alb-huKO boar yielded 7 non-Tg and 10 Tg pigs. Four of these Tg pigs carrying both of the transgenes exhibited both pancreas-specific Venus and liver-specific huKO expression in single individuals. Double-Tg pigs with pancreas-specific green fluorescence and liver-specific red fluorescence grew normally, and tests of their reproduction ability are currently underway. These data demonstrate that transgene introduction by ICSI-mediated gene transfer into in vitro-matured oocytes is a feasible option for generating pigs expressing fluorescent proteins in a tissue-specific manner.
33 PROPAGATION OF ELITE LIFESAVER DOGS BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Lee B. C. , Oh H. J. , Kim M. J. , Kim G. A. , Park E. J. , Choi J. , Yoo J. K. , Park J.-K. , Kim D.-H.
Published: December 2012

Canine somatic cell nuclear transfer (cSCNT) has been used as a useful tool for propagation of elite working dogs. In 2009, 7 cloned dogs were successfully produced using somatic cells derived from the excellent drug-sniffing dog of Korea Customs Service. All cloned dogs perfectly performed drug detection in Incheon International Airport. The objective of the present study was to compare the efficiency of the 2 activation culture media to clone the retired Baekdu, a veteran rescue dog that performed lifesaving activities worldwide for 6 years in Korea National Emergency Management Agency (NEMA). Ear tissue was collected from a 10-year-old male German Shepherd and fibroblasts were cultured for cSCNT. The cells were injected into the perivitelline space of enucleated in vivo-matured dog oocytes, fused with electric stimulation using an electro cell fusion apparatus (Nepa Gene Co. Ltd.), and activated chemically. In the activation protocol, 2 different types of media were tested to investigate the effect of proteins with undefined functions. The first medium was a modified synthetic oviduct fluid (mSOF), which is a complex culture medium with BSA that includes undefined functions. The second medium was the porcine zygote medium (PZM-5), which is a chemically defined medium with polyvinyl alcohol (PVA). The fused couplets were activated by mSOF medium supplemented with 1.9 nM DMAP (SOF-DMAP), and PZM-5 supplemented with 1.9 nM DMAP (PZM-DMAP) for 4 h, followed by 4 min of calcium ionophore treatment. Then, reconstructed oocytes were transferred into the uterine tube of naturally estrus-synchronized surrogate dogs. In the PZM-DMAP group, a total of 56 activated cloned embryos were transferred into 3 female recipient dogs, and a total of 64 activated cloned embryos from the SOF-DMAP group were transferred into 4 female recipients. Pregnancy diagnosis was performed using a SONOACE 9900 (Medison, Seoul, Korea) ultrasound scanner with 7.0-MHz linear-array probe between 30 and 35 days after embryo transfer. As a result, pregnancy was detected in 1 out of 3 surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and 1 pregnancy (25%) was detected in 4 surrogate mothers receiving cloned embryos from the SOF-DMAP group. Two pregnant dogs each gave birth to 1 healthy cloned puppy by cesarean section. This study shows that existence of proteins with undefined functions in activation medium did not affect the dog cloning. In addition, the number of elite working dogs in diverse fields can be increased by the NT technique using donor cells derived from small tissue of elite working dogs.
This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, and TS Corporation.
335 GENETIC ENGINEERING OF GOATS FOR THE PRODUCTION OF A BIOSIMILAR ANTIBODY IN MILK
Authors: Laible G. , Cole S.-A. E. , Brophy B. K. , Wright M. J. , Berg M. C. , Cullum A. A. , Delaney S. R. , Oback F. C. , Oliver J. E. , Pollock D. P. , Gavin W. G. , Wells D. N. , Meade H. M.
Published: December 2012

Dairy animals provide an attractive production platform for biosimilar antibodies due to the high protein production capacity of the mammary gland and easy access to milk. Goats are well suited for this approach as they offer a relatively short gestation time and good milk yield and are fully validated for the production of recombinant therapeutics. To generate transgenic goats capable of producing a biosimilar version of cetuximab, a monoclonal antibody for epidermal growth factor receptor and approved for the treatment of specific cancers, we co-transfected primary female fetal fibroblasts with expression constructs for cetuximab’s heavy (HC) and light (LC) chains under the control of the goat β-casein regulatory sequences. Beta-globin insulators were added to both transgenes to minimize position effects, and an antibiotic selection marker was placed downstream of the HC transgene sequences to allow for the isolation of stable transgenic cell clones. Selected cell clones were screened by PCR for the presence of both transgenes. Positive cell clones were analysed by Southern blot with a β-casein-specific probe. This allowed for the simultaneous detection of both transgenes, and the endogenous β-casein gene served as a standard to determine transgene copy numbers. The cell clones showed a broad range of copy numbers, from single copy insertions to >100 copies for the HC and LC transgenes. Interestingly, most of the cell clones had more LC than HC transgene copies. Ten cell clones were selected to generate transgenic founders using somatic cell nuclear transfer. We were able to produce 43 live kids from 9 cell lines following transfer of between 26 and 153 one- and two-cell embryos per line into recipients (range of 4 to 15 embryos per recipient). The one cell clone that we used unsuccessfully had the lowest number of transferred embryos (11). The efficiency for the production of live kids per transferred embryos was, on average, 5.1% (range of 1.0 to 9.7%). Kids from 5 lines were hormonally induced into lactation at the age of 10 weeks. Two lines with high copy numbers (≥30) produced either no or only a few drops of milk, whereas the lines with ≤25 transgene copies gave up to several milliliters of milk per day. Western analyses confirmed cetuximab production levels of 15 g L–1 in 2 of the lines with ≤25 transgene copies and ~45 g L–1 in a high copy number line; one low copy number line showed good HC but very low LC expression. Our data demonstrate that cetuximab can be produced in significant quantities in transgenic goats. Future work is aimed at determining production levels under natural lactation conditions and characterising glycosylation patterns to fully understand the pharmacodynamic properties of the antibody.
Supported by GTC, the NZ Ministry of Science and Innovation and AgResearch.
336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS
Authors: Koo B. , Kwon M. , Roh J. , Kim J. , Kim T.
Published: December 2012

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.
INSULATORS AND CHROMOSOME-TYPE VECTORS FOR GENE TRANSFER
Authors: Montoliu Lluis , Fernández Almudena , Seruggia Davide , Muñoz Diego , Vicente Cristina
Published: December 2012

Vertebrate genomes are functionally and structurally organised as gene expression domains. These domains contain all regulatory elements that are required for the gene (or genes) to be expressed correctly, according to a given pattern in time and space. The use of genomic-type DNA constructs in animal transgenesis ensures optimal transgene expression, once inserted into the host genome, because their large size include most if not all the regulatory elements that are needed for a given gene in order to be expressed correctly. Ideally, the entire gene expression domain is included. Large heterologous DNA molecules can be easily manipulated in bacterial or yeast cells, through the use of bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs), respectively. YACs are the vectors that allow the manipulation of the larger DNA molecules, in excess of 1 Mb (1000 kb). Some mammalian loci (i.e. the APP locus, ~450 kb) greatly exceed the maximum size for inserts that can be accommodated into BACs. Therefore, YACs are currently the only available robust and reliable solution to work with these large genes. The use of YACs, and/or BACs, usually ensures optimal transgene expression, because the heterologous constructs carry all that is required to resume the correct expression of the locus irrespective of the location where the transgene eventually lands in the host genome. Adjacent gene expression domains in genomes are often insulated by boundary elements, or insulators that insulate each domain and maintain the expression program of each gene according to their own set of regulatory elements, blocking any nondesirable interaction from its neighbouring locus. The function of these boundaries is, hence, to insulate gene expression domains in genomes allowing the protected locus to be expressed according to internal regulatory elements, without suffering from the adverse effects of flanking loci and without transmitting the effect of the internal regulatory elements beyond the protected domain. Insulators can act as enhancer blockers, preventing a distal enhancer from interacting with a given promoter, when placed in between, and/or as barriers, preventing the chromosomal position effects associated with random insertion of gene expression constructs in host genomes. In addition, insulators are known to contribute to the chromatin and nuclear structural organisation. A variety of molecular mechanisms has been associated with boundary function, probably reflecting the diversity of functional elements that can efficiently insulate genomic sequences. Insulator elements can be used in biotechnological applications, as spacers, as boundaries, in any gene expression construct to be used in gene transfer experiments, preventing the inappropriate expression patterns of transgenes and insulating them from neighbouring sequences surrounding the place of insertion in the host genomes.
51 INVESTIGATION OF INTER-INDIVIDUAL EPIGENETIC VARIABILITY IN BOVINE CLONES: A HIGH THROUGHPUT STUDY
Authors: Kiefer H. , Jouneau L. , Martin-Magniette M. L. , Balzergue S. , Campion E. , Chavatte-Palmer P. , Heyman Y. , Richard C. , Le Bourhis D. , Renard J. P. , Jammes H.
Published: December 2012

Reprogramming the differentiated cell to totipotency can be achieved following the introduction of its nucleus into an enucleated oocyte, a procedure known as cloning. We used cattle clones as a pertinent model to assess the inter-individual epigenetic variability and its consequences on phenotypes, including agronomically relevant traits and developmental pathologies. Indeed, the developmental defects frequently associated with cloning could be related to the insufficient extent of reprogramming, leading to perturbations of the nuclear microenvironment of the early embryo, with long-term consequences on the phenotype. Immunoprecipitation of methylated DNA following hybridization on a new bovine-specific tiling array (MeDIP-chip) was used to describe the epigenomic patterns affected by incomplete reprogramming. We first focused on the liver, because overgrowth of this organ is, to a certain extent, bound to global foetal overgrowth, which is often observed in clones. The microarray represents the 21 416 bovine genes currently annotated (UMD3.1 genome assembly). Each gene was tiled by 34 probes, on average, spanning upstream regions from –2000 to +1360 bp relative to the transcription start site. This microarray was hybridized with MeDIP samples from livers of normal Holstein animals obtained by AI (4 perinatal controls and 8 adults: 2 males and 10 females) and livers of female Holstein clones (7 perinatal clones from 2 different genotypes, either stillborn or suffering from severe pathologies, and 7 adult clones from 3 different genotypes, with normal to pathological phenotypes). After normalization of the data, enriched probes were identified using the ChIPmix method (Martin-Magniette et al. 2008 Bioinformatics) and located on the Ensembl Genome Browser. Results of exploratory analysis, including correlation clustering, principal components analysis (PCA), and independent components analysis (ICA), will be presented. A statistical test based on differences in the spatial distribution of the enriched probes along promoters was applied to the data, to associate epigenetic signatures to specific parameters (cloning, phenotype, stage, and genotype). Most promoters with more than 5 enriched probes across individuals showed a clustered distribution of the enriched probes. This local enrichment was highly conserved among individuals for 96% of the promoters, suggesting that most of the methylated regions were common to all animals. More interestingly, the distributions of the enriched probes showed inter-individual variability for 4% of the promoters in all samples. The identification and validation of these promoters is currently in progress.
Funding was provided by ANR-09-GENM-012-01 and ACI-PHASE-INRA-2010.
55 STUDY ON THE INTERSPECIFIC NUCLEAR TRANSFER OF PRZEWALSKI'S GAZELLES AND BOVINES
Authors: Gao Y. , Cheng L. , Su G. , Wei Z. , Li G.
Published: December 2012

Przewalski’s gazelle (Procapra przewalskii), also known as Platts antelope, is an endangered species only found in China. It belongs to the Artiodactyla order, Bovidae family, antelope subfamily, and Gazella genus. In this study, 5 experiments were designed to examine the developmental potential of Przewalski’s gazelle somatic cells transplanted into bovine enucleated oocytes. Enucleation was conducted by Hoechst 33342 staining of the oocytes and guided by a fluorescent microscope to ensure the removal of the nuclei. The gazelle cells were then transferred to the enucleated oocytes and electrically fused to reconstructed embryos. The study resulted in 5 major findings. (1) When gazelle-bovine reconstructed embryos were treated with the deacetylase inhibitor valproic acid (VPA), at different concentrations and for different times, treatment of the cloned embryos with VPA at 0.5 mM for 24 h significantly increased the 8- to 16-cell-stage embryo development [61.9% (96/155) v. 33.8% (46/136) control]. However, the morula [1.3% (2/155) v. 1.5% (2/155); P > 0.05] and blastocyst (0.7% v. 1.5%; P > 0.05) development were similar to that of the control. In the intraspecific (bovine-bovine) control group, the cleavage, morula and blastocyst development of 3 cloned embryos were 72.6% (127/175), 28.0% (49/175), and 23.4% (41/175). (2) Octamer-binding transcription factor 4 (Oct-4), as a developmental potential and expression marker, was transfected to gazelle cells. When Oct-4-eGFP-confected cells were transferred, the cloned embryo development did not improve either with or without VPA treatment. (3) When the gazelle-bovine embryos were treated with the deacetylase inhibitor trichostatin A (TSA) for 24 h at 10 ng mL–1, blastocyst development was significantly higher than in the control group [3.6% (6/168) v. 0.8% (1/125); P < 0.05]. (4) When a reverse NT protocol, in which the oocyte nucleus was removed after the cell nucleus was fused to the oocyte, was used for NT, the cloned embryo development did not improve. (5) The gazelle-bovine and bovine-bovine cloned embryos at 8- to 16-cell stages, gazelle cells, bovine cells, and bovine oocytes transcriptomes were analyzed by Affymetrix microarray (Affymetrix Microarray Inc., Santa Clara, CA, USA) and repeated twice. A total of 643 genes were activated in gazelle-cattle embryos compared with oocytes, whereas 1527 genes were activated in bovine-bovine clones. A total of 1010 genes that were exclusively expressed in gazelle somatic cells were still expressed in the interspecies cloned embryos. In conclusion, TSA treatment of Przewalski’s gazelle somatic cells transferred into enucleated bovine oocytes improved development of cloned embryos to the blastocyst stage, although still with low efficiency. Data from microarray analyses of the gazelle-cattle embryos showed that over 1000 gazelle-specific genes were still expressed in the interspecific cloned embryos.

This work was supported by the National Basic Research Program of China (no. 2012CB22306).