bluuebunny/crossref_metadata_2025 huggingface dataset:
295 EQUINE AMNION-DERIVED MESENCHYMAL STEM CELLS: POSSIBLE IMPLICATION IN ENDOMETRIAL REGENERATION
Authors: Corradetti B. , Perego R. , Meucci A. , Bizzaro D. , Cremonesi F. , Lange-Consiglio A.
Published: December 2012

In veterinary medicine, as in human, amnion is an attractive source of mesenchymal stem cells (MSC), which poses no ethical dilemmas, allows highly efficient recovery of cells without the requirement of invasive procedures and shows immunomodulatory properties. We previously demonstrated that equine amniotic membrane-derived cells (AMC) not only exhibit specific stem cell properties with respect to expression of pluripotent (OCT-4, TRA-1-60, and SSEA-4) and adult (CD44, CD105, CD29, and CD166) stem cell markers but also possess differentiation potential in vitro and the capability to regenerate tendons in vivo after spontaneous lesions when allogeneically transplanted. Moreover, we reported evidence that at the first passages (P) in culture (until P5), AMC express MHC-class I but not MHC-class II and are well tolerated in vivo. In the present study, we further characterised AMC in vitro in order to evaluate their potential application in the treatment of endometritis in vivo. In particular, the amniotic membrane in toto and AMC have been compared to the endometrial tissue in toto and to cells isolated from endometrium for the expression of genes involved in the proliferation and differentiation of uterine MSC during early pregnancy (AbdB-like Hoxa genes) and those influencing preimplantation conceptus development (progesterone receptor, PR, and oestrogen receptors ERα and ERβ). The AMC were isolated as recently reported by Lange-Consiglio et al. (2011 Open J. Tissue Eng. Regen. Med. 4), and endometrial cells were obtained according to the protocol described by Donofrio et al. (2008 Reprod. Biol. Endocrinol. 6, 65) for bovine cells, and slightly modified for equine cells. Total RNA was extracted from both tissues and from AMC and endometrium-derived cells immediately after isolation (P0). Reverse transcription-PCR was performed according to the standard procedures, using GAPDH and HPRT1 as reference genes. Expression of HOXA9 and PR was confirmed in all samples examined, whereas mRNA for ERβ was only detected in endometrial tissue and in cells derived from it. Expression of ERα was observed only in endometrial tissue. The expression of genes crucially involved in patterning of the female reproductive tract (HOXA9 and PR) in amnion and cells derived from it suggests that this source shares similar molecular properties with endometrium. Further studies are required to explore uterine mesenchymal-like features shared by AMC (i.e. verifying the expression of Wnt7α, Wnt5α, and Wnt4α, or the presence of the more recently characterised membrane-bound intracellular progesterone receptors PGRMC1 and mPR). These preliminary results provide an intriguing indication of the possible implication of amnion-derived cells in endometrial regeneration, in particular when poor endometrial proliferation is associated with infertility or poor pregnancy outcome.
306 ANTITUMOR EFFECT OF ENDOMETRIAL CANCER MASS WAS INDUCED BY THERAPEUTIC STEM CELLS FUSED CYTOSINE DEAMINASE AND INTERFERON-BETA IN A NUDE MOUSE MODEL
Authors: Yi B.-R. , Choi K.-C.
Published: December 2012

Endometrial cancer has been reported as the most common malignancy, accounting for ~50% of the uterine tumour cases in the Western world. An exact mechanism underlying the development of this type of cancer is unclear. In our previous study, we confirmed the therapeutic efficacy of stem cells expressing bacterial cytosine deaminase (CD) and/or human interferon-β (IFN-b) gene against endometrial Ishikawa cells in an in vitro model. The CD gene can convert a nontoxic prodrug, 5-fluorocytosine (5-FC), to the toxic agent, 5-fluorouracil (5-FU), and IFN-b is a powerful cytokine that has antiviral effects. In this study, we generated xenografted mice baring Ishikawa endometrial cancer cells and confirmed the therapeutic efficacy of these stem cells. For generation of an animal model of endometrial cancer, Ishikawa cells (2 × 106 cells/mouse) were implanted subcutaneously in severe combined immunodeficiency (SCID) mice. Next, the stem cells stained CM-DiI as red fluorescence were injected into nearby tumour mass and, 1 day after stem cell injection, 5-FC (500 mg kg–1 per day) was adminstered through an intraperitoneal injection per day for 14 days. Two days after final injection of the prodrug, we killed these mice and extracted the endometrial tumour mass. Tumour volumes reached 1400 and 450 mm3 in control mice and mice injected with stem cells, respectively, and fused gene expression of CD and/or IFN-b significantly inhibited the growth of endometrial cancer mass in the presence of 5-FC (~50 to 60%). In addition, we confirmed the migratory effect of the stem cells using a fluorescent analysis; red-stained HB1.F3.CD and HB1.F3.CD.IFN-b cells were found in the extracted endometrial cancer mass. A histological analysis of tumour mass showed that the aggressive property of endometrial cancer was inhibited in the presence of a prodrug in mice treated with therapeutic stem cells. Taken together, these results provide evidence for the usefulness of therapeutic stem cell-based gene therapy through a targeted delivery of therapeutic gene products to endometrial cancer sites.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) of Korea government (no. 2010-0003093).
313 EFFECT OF DURATION OF THE GROWING PHASE OF OVULATORY FOLLICLES IN SUPERSTIMULATED HEIFERS ON OOCYTE COMPETENCE AFTER IN VITRO FERTILIZATION
Authors: Dias F. C. F. , Dadarwal D. , Honparkhe M. , Adams G. P. , Mapletoft R. J. , Singh J.
Published: December 2012

We tested the hypotheses that extending the duration of follicular growth by superstimulation increases oocyte competence, and that FSH starvation at the end of superstimulatory treatment decreases oocyte competence. Heifers were allocated randomly to short FSH duration (n = 8), FSH starvation (n = 8), or long FSH duration (n = 8) groups. Five to 8 days after ovulation, transvaginal ultrasound-guided follicle ablation was done to synchronize follicle wave emergence, and a progesterone-releasing device (CIDR; Pfizer Animal Health, New York, NY, USA) was placed intravaginally. Short FSH and FSH starvation groups were given 8 doses of FSH (Folltropin-V; Bioniche Animal Health Inc., Belleville, ON, Canada) IM, whereas the long FSH group was given 14 doses of FSH at 12-h intervals, starting from the day of wave emergence (Day 0). Prostaglandin F2α (PGF) was administered twice, 12 h apart, on Day 3 in the short FSH group and on Day 6 in the other 2 groups. In all heifers, the CIDR was removed at the time of the second PGF treatment; pLH (Lutropin-V; Bioniche Animal Health Inc.) was given IM 24 h after CIDR removal, and cumulus–oocyte complexes (COC) were collected 24 h after pLH treatment. The COC were matured in vitro (6 h) and fertilized (IVF), and the embryos were cultured for 10 days. At 12 h after pLH, the long FSH group had a greater number of ≥9 mm follicles than the FSH starvation and short FSH groups (25.4 ± 5.3 v. 11.0 ± 2.1 and 10.6 ± 2.3, respectively; P < 0.03). The long FSH group also had more expanded COC than the FSH starvation group (P < 0.001), but did not differ from the short FSH group (93, 54, and 74%, respectively). The FSH starvation group had a greater proportion (P < 0.0001) of partially expanded COC (32%) and poor quality oocytes (70%) than did the long (1 and 33%) and short (4 and 45%) FSH groups; oocyte quality did not differ between long and short FSH groups. At 48 h after IVF, the cleavage rate was lower in the FSH starvation group compared with the short and long FSH groups (35, 54, and 56%, respectively; P = 0.003). After 9 days in culture, embryo development (morula + blastocyst) in the FSH starvation group was lower than that in the long FSH group, (18 v. 37%; P = 0.04), but did not differ from that in the short FSH group (25%). After removal of the data of one heifer in the FSH starvation group that produced 52% of total embryos in that group (outlier), the Day 9 blastocyst rate was lower in the FSH starvation group than in the short and long FSH groups (2% v. 14 and 21%, respectively; P = 0.02). In conclusion, extending the standard superstimulation protocol by 3 days enhanced ovarian response to FSH treatment, but did not improve oocyte competence, whereas a period of FSH starvation after FSH treatment compromised oocyte quality and embryo development.
323 PRODUCTION OF Mx2-TRANSGENIC PIGS FOR INFLUENZA STUDY
Authors: Jeon Y. , Jung E. M. , Kim Y. K. , Kwak S. S. , Cheong S. A. , Jeung E. B. , Lee E. , Kim N. H. , Hyun S. H.
Published: December 2012

A worldwide flu pandemic occurred in 2009, resulting in many victims and high social damages. In the A (H1N1) virus spreading process, the pig is the intermediate host, and this virus is amplified and genetically changed through recombination in pigs. The objective of this study was to develop influenza-resistant pigs. In interferon-α and interferon-γ treated cells, the porcine Mx2 protein has been observed near the nuclear envelope, which consequently has been linked with inhibition of influenza virus proliferation. Therefore, we attempted to produce transgenic (TG) pigs overexpressing the Mx2 gene by somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were transfected with the cytomegalovirus vector, which includes the porcine Mx2 gene. The established transgenic cell was injected into the enucleated ooplasm to produce Mx2-TG cloned embryos. In total, 511 female TG porcine embryos were transferred to 5 surrogates. Two recipients were diagnosed pregnant (pregnancy rate, 40%) on Day 25. On Day 114, 6 fetuses and 4 mummies were collected. The PCR analysis concluded that there was no integration of the Mx2 gene. Then, a male Mx2-TG cell line was established to use as donor cell of SCNT. In total, 547 male TG-SCNT embryos were produced. Of these, 38 embryos were cultured in vitro to confirm the developmental capacity of the embryos. Among these porcine SCNT-TG embryos, 26 embryos (68.4%) cleaved and 5 (13.2%) developed to the blastocyst stage. The PCR analysis confirmed that all male TG-SCNT blastocysts were for integration of the Mx2 gene. The remaining 509 male embryos were transferred to 5 surrogates. Two recipients (pregnancy rate, 40%) were diagnosed pregnant at Day 25. To date, 1 of the surrogate has maintained pregnancy and another recipient gave birth to 9 piglets. Two days after birth, 2 piglets died and the remaining piglets remain healthy. Verification analysis of gene targeting and resistance to influenza is in progress. This study has presented new possibilities of production of influenza virus resistant pig by SCNT for translational research.
This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121012011), Rural Development Administration, Republic of Korea.
324 PRODUCTION OF GENETICALLY MODIFIED PIGS BY ARTIFICIAL REPRODUCTIVE TECHNOLOGIES USING FROZEN EPIDIDYMAL SPERM
Authors: Honda K. , Takeuchi Y. , Matsuda T. , Kanai T. , Kuramoto M. , Maehara M. , Matsunari H. , Nakano K. , Umeyama K. , Watanabe M. , Nakauchi H. , Nagashima H.
Published: December 2012

Genetically modified (GM) pigs are useful tools for many types of biomedical research. The objective of this study was to develop a reliable protocol for the reproduction of GM pigs using frozen epididymal sperm. Epididymal sperm were collected from 3 GM boars: (1) humanized Kusabira-Orange (huKO) transgenic, (2) mutant human hepatocyte nuclear factor 1α (HNF-1α) transgenic, and (3) α1,3-galactosyltransferase gene knockout (GalT-KO). Additionally, a wild type (WT) boar was also used for sperm collection. After collection, the sperm were frozen in Niwa and Sasaki freezing medium (Niwa et al. 1989 Manual for Cryopreservation of Pig Spermatozoa 19–23; 1.0 × 109 sperm mL–1) using 0.5-mL straws. The sperm were cooled utilising the 2-step method, from 25 to 15°C at a rate of 0.17°C min–1, and then from 15 to 5°C at 0.08°C min–1. After cooling to 5°C, the straws were frozen in liquid nitrogen with 3% glycerol for storage. After storage for 0.5 to 21 months, the straws were thawed by immersion into a 37°C water bath. The recovered sperm were washed by centrifugation in PBS + 1 mg mL–1 BSA and were resuspended in either Beltsville thawing solution (Pursel and Johnson. 1975 J. Anim. Sci. 40, 99–102) for intra-fallopian AI or in porcine fertilization medium (PFM, Research Institute for the Functional Peptides, Yamagata, Japan) for IVF. Prepubertal gilts (6.5 months), in which oestrus was induced by 1000 IU eCG and 1500 IU hCG given 66 or 72 h apart, were surgically inseminated with 0.9–7.9 × 106 progressive motile sperm 41 to 51 h after receiving an hCG injection. Oocytes, which matured in vitro either in modified porcine oocyte medium (IFP) or modified NCSU23 medium, were inseminated in PFM for 8 h with either 0.5 (huKO, GalT-KO) or 1.6 (HNF-1α) × 106 progressive motile sperm mL–1. The in vitro fertilized oocytes were cultured in porcine zygote medium-5 (Research Institute for the Functional Peptides) for 5 to 6 days until they were transferred. Some of these oocytes were examined for their fertilization rates. Blastocysts were transferred into the uterus of the recipient 150 h after hCG treatment. The fertilization rates for huKO, HNF-1α and GalT-KO sperm were 63.5% (61/96), 62.5% (70/112) and 92.4% (61/66), respectively; 50.8% (31/61), 82.9% (58/70) and 60.7% (37/61) of these were monospermic. The rate at which the IVF embryos developed into blastocysts was 47.8–52.4%. This study demonstrates that IVF using frozen epididymal sperm is more efficient method for reproducing genetically modified pigs than AI (87.5 v. 33.3%).

Table 1.Pig production efficiencies of frozen epididymal sperm from genetically modified pigs utilising AI and IVF

This study was supported by JST, ERATO, NAKAUCHI Stem Cell and Organ Regeneration Project, Tokyo.
325 PRODUCTION OF GAL KNOCKOUT/hA20 TRANSGENIC PIGS WITH IMPROVED XENOPROTECTIVE PROPERTIES
Authors: Ahrens H. E. , Petersen B. , Petkov S. , Hauschild-Quintern J. , Niemann H.
Published: December 2012

Pig-to-human xenotransplantation is promising for overcoming the shortage of suitable human donor organs, but is hampered by immunological barriers. The next immunological hurdle is the acute vascular rejection (AVR), which is associated with activation of the endothelium and the coagulation system. Recently, we demonstrated that transgenic expression of the zinc finger protein A20 protects porcine cells against apoptotic and inflammatory stimuli (Oropeza et al. 2009 Xenotransplantation 16, 522–534). Compared with other anti-apoptotic proteins, A20 also has immune-modulatory potential as shown in an CD95(Fas)Ligand assay. However, in that study, hA20 was only expressed in skeletal muscle, heart and porcine aortic endothelial cells of transgenic pigs. For use in xenotransplantation, it is critical to produce pigs with ubiquitous expression of hA20. Here, we constructed a new vector based on the Sleeping Beauty transposon plasmid pT2/HB containing the hA20 cDNA driven by the ubiquitously and strongly expressing CAGGS-promoter and co-transfected gal–/– porcine fibroblasts (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010) together with the SB transposase 100X plasmid (pT2/HB and SB transposase both kindly provided by Dr. Zoltan Ivics). Cells were selected with 400 µg of G418/mL of medium for 14 days. Subsequently, cells were screened by PCR. Transfected cell clones were pooled and used as donor cells in somatic cell nuclear transfer. Reconstructed embryos were transferred to 2 synchronized sows. Both remained pregnant on Day 25 of gestation. One recipient is expected to deliver in September 2012. The second sow, which received 104 embryos, was sacrificed on Day 26 of pregnancy and 2 fetuses could be obtained (cloning efficiency 1.92%). The fetuses had integrated the transgene into their genome as shown by PCR. Real-time PCR results from fetal fibroblasts indicated similar hA20 mRNA expression levels in both fetuses, whereas wild type controls were negative. The hA20 expression level was 2.5 and 3.1 times lower than in the original pooled of transfected cells. Fetus #1, which showed a slightly higher hA20 mRNA expression, was used for recloning. In total, 3 more recipients received an average of 104 hA20 transgenic embryos each. Currently, the hA20 protein level in fetal fibroblasts is determined by fluorescence activated cell sorting analysis. Once pigs are born, the tissue distribution of hA20 will be analysed. In parallel, the function of the transgene will be studied in the CD95(Fas)Ligand assay. Hearts and kidneys of the hA20-transgenic pigs will be further tested in ex vivo perfusion assays and in a pig-to-baboon xenotransplantation. This approach is promising for advancing pig-to-human xenotransplantation to preclinical application.
328 VERSATILE TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE (TALEN)-MEDIATED ENGINEERING OF OSSABAW MINIATURE SWINE
Authors: Carlson D. F. , Tan W. , Fahrenkrug S. C.
Published: December 2012

Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA-binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. TALEN-induced double-strand breaks can be used for gene inactivation via repair by nonhomologous end joining (NHEJ) or to stimulate homologous recombination with a user-specified template to create custom-designed disease alleles. Transfection of multiple TALEN pairs targeting the same chromosome can also produce chromosomal rearrangements such as large inversions and deletions. As genetic modification of primary pig fibroblast followed by cloning is the primary method used to generate transgenic swine, we tested our capacity to mimic multiple common mutation types in primary fibroblasts using TALEN. Either plasmids or mRNA encoding TALEN was transfected into 500 000 to 750 000 fibroblasts by nucleofection followed by 3 days of culture at 30°C before analysis of activity. With this approach, we found that over 65% of custom-designed TALEN displayed activity in pig fibroblasts with typical percentage of indel positive chromosomes ranging from 20 to 45%. Individual clones were derived by either co-selection with an unlinked selection transposon or by dilution cloning. Colonies with mono- and biallelic modification to the target loci were readily identified (up to 84 and 24%, respectively) and could be accomplished without the aid of selection. Co-transfection of 2 TALEN pairs directed to the same chromosome resulted in deletion or inversion of the intervening sequence in 10 and 4% of isolated colonies, respectively. In addition, co-transfection of TALEN with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, 3% of which were homozygous (n = 184 colonies). To prove that modified cells were suitable for generation of animals, we pooled several colonies of Ossabaw fibroblasts harboring TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for cloning. Pregnancy was established in 7/9 transfers (100–150 embryos per transfer), and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Three monoallelic frame-shift genotypes were observed among 9 piglets; as expected, each genotype was represented in the parental cell pool. The remaining 9 animals displayed an identical biallelic mutant genotype and are being investigated as a knockout model of familial hypercholesterolemia (FH). Finally, fibroblasts homozygous for a specified inactivating mutation were created by allele introgression and were cloned, demonstrating that gene editing can be used to create precise swine knock-ins in a single generation.
329 FLOW CYTOMETRIC ANALYSIS OF SPERMATOZOA FROM REPORTER TRANSGENIC BOARS DERIVED BY PRECISION GENETIC ENGINEERING
Authors: Garrels W. , Holler S. , Cleve N. , Klein S. , Ivics Z. , Niemann H. , Rath D. , Kues W. A.
Published: December 2012

Recently, we produced 2 founder boars with a non-autonomous Sleeping Beauty (SB) system carrying 3 monomeric integrations of a Venus transposon cassette and showing transgene segregation during meiosis (Garrels et al. 2011 PLoS One 6, e27563). It was possible to show transmission of the reporter protein to fertilized oocytes by confocal microscopy. The aim of this study was to assess the suitability of different fluorophore reporters for in vivo labelling of pig spermatozoa. Therefore, we used Venus transposon fibroblasts from a F1 boar, which carry a single integration of the transposon cassette and used these fibroblasts for a Cre-mediated cassette exchange against an mCherry reporter. These cells were used for somatic cell nuclear transfer (SCNT) to derive a syngene clone cohort of boars, which differ only in the fluorophore reporter cDNAs (either Venus or mCherry). Importantly, this methodology did not request any antibiotic selection cassette and allows precise genetic modifications in a livestock species where no authentic embryonic stem cells are available (Garrels et al. 2012 Trends in Biotechnology 30, 386–393). A total of 8 male piglets carrying the Venus transposon, and 4 male piglets carrying the mCherry reporter were born. Three Venus boars and 2 mCherry boars were raised to sexual maturity, and ejaculated sperm was obtained with the help of a phantom. A detailed flow cytometric analysis revealed that the spermatozoa samples were specifically Venus or mCherry positive [Gallios, Beckmann Coulter, Krefeld, Germany; solid-state laser (488 nm; 22 mW), filter for green fluorescence (525 BP); filter for red fluorescence: (620/30)], respectively. In direct comparative measurements, the spermatozoa samples from transgenic boars (Venus and Cherry) and wildtype controls could be discriminated. Interestingly, spermatozoa were uniformly Venus- or mCherry-positive and gave a distinct fluorescence peak in flow-cytometric measurements. The monomeric transgenes were transmitted through the germ line according to Mendelian rules with the expected ratio of 50% transgenic and 50% nontransgenic piglets. Fluorescence microscopic analysis and Western blotting confirmed the uniform presence of Venus and mCherry in boar spermatozoa, respectively. This is the first characterisation of spermatozoa from a pig cohort carrying a targeted cassette exchange. This large animal model may help to elucidate the function of paternally transmitted components to fertilized oocytes.
VIRAL AND NONVIRAL VECTORS
Authors: Whitelaw Bruce
Published: December 2012

Genetically engineered (GE) livestock have existed since the mid-1980s, and, since then, a range of methods for delivery of the transgene have been developed, each with advantages and limitations. In regards to the wealth of possible methods for the production of GE animals, two general approaches have emerged. Historically first, the direct manipulation of the zygote (including manipulation of germ cells) now comes in many flavours, from direct pronuclear injection of double-stranded DNA constructs to the use of vectors to deliver the transgene. The second approach utilises an in vitro stage where cells are engineered in culture before being introduced in some way to the developing embryo. The former suffers from lack of control of transgene integration, which can expose the transgene to position effects. The latter can be exploited through homologous recombination to engineer specific genetic loci; with somatic cell cloning being the most widely used method. Both approaches can now be combined with the exciting new editor technologies to enable precise genome editing which in some cases does not involve the incorporation of a transgene. For methods involving the zygote, the use of specific vectors can be of advantage, and the same can be true for the manipulation of cells; however, many delivery strategies are possible for this process. Overall, the drivers for delivery method development have revolved around efficiency and specificity. With regard to viral vectors, and possibly nonviral nanoparticle formulations in the future, spectacular increases in transgenesis rates can be achieved. With the most widely used vector, based on a lentivirus genome, in some cases all animals born from injected zygotes can be transgenic. In livestock, where gestation and breeding times are long, this dramatically reduces the time to proof-of-concept for a given project. In addition, these founder animals will carry different transgene copy-numbers, which is associated with different levels of transgene expression. This strategy can be exploited to quickly produce a large cohort of animals that enable modelling of the range of phenotype observed in a population for a given disease. In addition to the delivery of a transgene, such vectors can also be beneficial for the delivery of reagents that facilitate genome engineering, the most exciting of which are the genome editors. In this situation, either the editor and/or any DNA sequence to be incorporated can be efficiently delivered if not essential for broad uptake of this approach. It is likely that nonintegrating vectors will be desirable. In summary, viral vectors have a broad utility in facilitating the production of GE animals. In the future, nonviral nanoparticles may offer similar opportunities. Given the breadth of methodologies available and with the anticipated use of GE livestock in both agricultural and biomedical applications gaining momentum, we are entering an era of unparalleled opportunity in this area of animal biotechnology.
DISEASE RESISTANCE AND OTHER APPLICATIONS OF TRANSGENESIS IN THE CHICKEN
Authors: Sang Helen
Published: December 2012

Genetic modification of the chicken in terms of gene addition is now robust and efficient. Transgenes can be introduced by injection of lentiviral vectors into chick embryos or by transfection of transposon vectors into embryos or primordial germ cells in vitro. Lentiviral vectors are limited in the size of transgene they can incorporate but we have generated several different transgenic lines using HIV-derived vectors and have observed high levels of transgene expression and tissue-specific expression using regulatory sequences from several genes. M. McGrew (The Roslin Institute) has established primordial germ cell lines and effective methods for transfection with piggyBac and Tol2 transposon vectors. The primordial cells are injected into chick embryos where they populate the developing gonads and contribute to the germline in mature birds. The availability of primordial germ cell lines will also form the basis of using artificial site-specific nucleases for gene knockout and potentially gene targeting in the chicken. These technologies facilitate the application of transgenesis in the chicken for basic research and for potential applications in poultry breeding. The chick embryo is an invaluable model for studying vertebrate development as the embryos can be accessed in ovo or in culture at the earlier stages of development. Embryos can be transfected with transgenes by electroporation and manipulated to study many aspects of development. We are developing transgenic chickens in which fluorescent protein reporters are expressed either ubiquitously or in targeted cell types. These form the basis of novel tools for increasing the value of the chick embryo in studying development. We provide fertile eggs from these lines to other research groups and are investigating the development of macrophages using a macrophage-targeted reporter. The potential for the use of genetic modification to be used in poultry breeding can now be explored. Commercial poultry production is challenged by several major pathogens including avian influenza. Flocks can be protected by good biosecurity measures and/or vaccination but vaccination is not always effective. It may be possible to add novel genes to the chicken genome targeting avian influenza virus replication. We are developing this approach (with L. Tiley, Cambridge University) and have generated transgenic chickens that do not transmit avian influenza when directly infected with H5N1 virus.
37 THE EFFECTS OF A CLASS III HISTONE DEACETYLASE INHIBITOR (SIRTINOL) ON EARLY DEVELOPMENT OF PORCINE CLONED EMBRYOS
Authors: Kwak S.-S. , Jeon Y. , Yoon J. D. , Cheong S.-A. , Lee E. , Hyun S.-H.
Published: December 2012

Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100 µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P < 0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12 h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200 µM sirtinol treatment for 12 h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P < 0.05) improved by treatment with 150 µM sirtinol for 12 h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200 µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150 µM sirtinol treatment for 12 h after activation with or without 5 nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality.
This work was supported by a grant from the Next-Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.
42 EFFECT OF S-ADENOSYLHOMOCYSTEINE, A NON-TOXIC EPIGENETIC MODIFYING REAGENT, ON PORCINE FEMALE DONOR CELLS AND CLONED EMBRYOS
Authors: Kang J. T. , Choi J. Y. , Park S. J. , Kim S. J. , Moon J. H. , Jang G. , Lee B. C.
Published: December 2012

Despite great advances in the field of cloning techniques, the efficiency of production of cloning animals is very low. Maybe the poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell or cloned embryos. The objective of this study was to investigate the effects of treatment with S-adenosylhomocysteine (SAH), the reversible nontoxic inhibitor of DNA methyltransferases (DNMT), on porcine female fibroblast donor cells and in vitro development of cloned embryos. We hypothesized that SAH targeting DNA methylation could alter chromatin configuration and turn it more amenable to reprogramming. Thus, the female fibroblast donor cells were cultured in media containing respective concentrations of SAH [0 (control), 0.1, 0.5, and 1 mM) for 2 passages. One-way ANOVA was used to determine significant differences in the data and a Tukey test was done to determine statistical differences among groups. Compared with nontreated controls, the cells treated with SAH, especially 1 mM, revealed significantly (P < 0.05) reduced global DNA methylation, proved by commercial kit and immunocytochemistry analysis, and elevation of transcript levels for X chromosome-linked genes (XIST and HPRT), estimated by real-time PCR analysis compared with the control group. It was suggested that treatment with SAH in female cells could make cells into more valuable donor cells for cloning. In another trial, cloned embryos using normal donor cells were cultured in media containing 1 mM SAH for 0 (control), 12, and 24 h after activation on different time interval of DNMT inhibition, transferred to PZM5 media, and subsequently cultured for 7 days. Treatment with SAH for 12 h resulted in 13.0 ± 1.9% blastocyst production, which was significantly greater than cloned embryos treated with SAH for 24 h (11.2 ± 2.1%) and control cloned embryos (9.1 ± 1.2%). It was suggested that the appropriate DNMT inhibition might have an important role in in vitro development of porcine SCNT, and improving effects on developmental competency of cloned embryos. We concluded that SAH induced global DNA demethylation that partially reactivated the X chromosome and that a hypomethylated genome may facilitate the nuclear reprogramming process.
This study was supported by IPET (no. 311011-05-1-SB010), MKE (no. 10033839-2012-21), Institute for Veterinary Science, the BK21 program, and TS Corporation.
44 LIMITATIONS OF PORCINE OOPLASM TO REPROGRAM BOVINE SOMATIC CELLS
Authors: Strejcek F. , Ostrup O. , Morovic M. , Petrovicova I. , Lucas-Hahn A. , Petersen B. , Niemann H. , Hyttel P. , Laurincik J.
Published: December 2012

The oocyte cytoplasm (ooplasm) constitutes a unique mixture of factors that are critical for successful reprogramming of haploid maternal and paternal genomes at fertilization as well as the diploid somatic cell genome after somatic cell nuclear transfer (SCNT). The 1-cell-stage embryos (produced by transfer of bovine fibroblast into porcine enucleated ooplast; interspecies SCNT, iSCNT) were processed at different time points (2, 4, 8, and 12 h post-activation, hpa) for detailed nuclear and nucleolar analysis by transmission electron microscopy (TEM; 4 to 5 embryos per group), and immunofluorescence (4 and 12 hpa) for visualization of nucleolar proteins related to transcription (upstream binding factor, UBF) and processing (fibrillarin; 8 to 9 embryos per group). The parameters of interspecies embryos were compared with porcine parthenogenetic (PA) counterparts (4 to 5 embryos per group for TEM analysis; 8 to 9 embryos per group for immunofluorescence analysis). At all evaluated time points, embryos in both groups displayed pronucleus-like nuclei with abundant euchromatin and characteristic porcine nucleolus precursor body (NPB), indicating maternal origin of nucleolar components. Fibrillarin was in both groups localized into shell-like intranuclear entities surrounding NPB. On the contrary, UBF in PA embryos was at 12 hpa colocalized with fibrillarin, whereas in iSCNT embryos, UBF staining was absent at both time points. Despite the similar morphology and localization of processing factor fibrillarin, striking differences in localization of transcription factor UBF suggest limitation of porcine ooplasm to mediate initial phases of nucleolar remodelling.
This work was supported by VEGA 1/0077/11.
47 DIFFERENCES IN MITOCHONDRIAL DNA COPY NUMBER AND EPIGENETIC PATTERNS OF MITOCHONDRIA-RELATED GENES IN CLONED COWS FROM THE SAME DONOR CELLS
Authors: Kaneda M. , Watanabe S. , Hirao Y. , Akagi S. , Haraguchi S. , Somfai T. , Takeda K. , Hirako M. , Geshi M. , Nagai T.
Published: December 2012

The genetic codes of cloned animals and the donor are identical; however, incomplete reprogramming of donor nuclei during NT causes epigenetic abnormalities in cloned animals. Due to the genetic identity and epigenetic differences among clones, we can study epigenetic effects on the phenotypes by analyzing genetically identical clones. During the NT process, donor cell mitochondria (mt) are transferred into the recipient oocytes and mtDNA heteroplasmy is observed. Previous studies have reported various mtDNA transmission patterns not only in the cloned animal itself but also in the offspring of clones. However, differences in mtDNA copy number in cloned animals have not been reported, especially genetically identical ones. To analyze the genetic effects on mtDNA copy number in cattle, we compared actual mtDNA copy number per diploid genome in various tissues of clones derived from the same donor cells. From 5 genetically identical cloned cows (Japanese Black cattle, ages 68 to 82 months) and 6 non-cloned cows (Japanese Black cattle, ages 52 to 129 months), we isolated DNA from 8 kinds of tissues (heart, lung, liver, kidney, spleen, small intestine, muscle, and spinal cord) and measured mtDNA copy number by using real-time PCR. The absolute copy numbers of 2 mtDNA-encoded genes (COX1 and CytB) and 2 nuclear-encoded genes (H19 and IGF2) were measured and analyzed. To examine the epigenetic effects on mitochondria-related genes, we also analyzed DNA methylation patterns of mitochondria-related gene ANT4 (mitochondrial ADP-ATP translocase) in these tissues by the combined bisulfite restriction analysis (COBRA) method. The actual mtDNA copy number per diploid genome varied in tissues and individuals both in clones and non-clones (average in clones v. non-clones: heart: 11 839 ± 6210 v. 9569 ± 2555; lung: 2027 ± 1153 v. 1383 ± 173; liver: 5644 ± 2278 v. 4799 ± 1848; spleen: 1080 ± 844 v. 393 ± 265; kidney: 7034 ± 4448 v. 2939 ± 784; small intestine: 1330 ± 573 v. 437 ± 171; muscle: 9861 ± 3640 v. 7907 ± 3229; spinal cord: 3961 ± 1819 v. 2756 ± 496). The variability of mtDNA copy number in clones was significantly higher in the lung, spleen, kidney, small intestine, and spinal cord (P = 0.001, 0.026, 0.005, 0.021, and 0.014, respectively; F-test), but not in other tissues. Methylation of the ANT4 gene is quite tissue dependent: hypomethylated in the liver, muscle and spinal cord; moderately methylated in the heart, lung, and kidney; and highly methylated in the spleen and small intestine. The methylation patters of ANT4 were not different between clones and non-clones. These results suggest that mtDNA copy number is more influenced by nongenetic factors than genetic background.
50 EFFECTS OF REPROGRAMMING TRANSCRIPTION FACTORS AND CHEMICAL INHIBITORS ON THE DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Yang B.-C. , An J.-H. , Chung H. K. , Jo S.-J. , Chung H.-J. , Kim K.-W. , Han D.-W. , Lee S.-S. , Yoo J.-K. , Park J.-K.
Published: December 2012

Currently, somatic cell nuclear transfer (SCNT) is regarded as the most efficient technique to produce transgenic cattle. However, compared with other techniques, such as IVF and in vivo embryo, the birth rate of SCNT embryos has been disappointingly low. Because incomplete epigenetic reprogramming in SCNT embryos has been suggested as a major reason for this low birth rate, we attempted to improve the success rate by using several lines of donor cells that were reprogrammed by gene transfection and an inhibitor treatment. First, the bovine ear fibroblast cells were transfected with vectors (pCX-OKS-2A and pCX-Myc) carrying 4 genes (Oct-4, Klf4, Sox2, and c-Myc; OKSM). Subsequently, transfected cells were incubated in the presence of one of the following chemical inhibitors: (1) trichostatin A (TSA, 20 nM), (2) 5-aza-20-deoxycytidine (5-aza-dC, 10 µM), or (3) GSK-3/MEK inhibitor (2i; GSK-3, 3 µM and MEK, 1 µM) for 7 days. Throughout the incubation period, cells in each treatment group were analyzed for their histone acetylation status. Subsequently, transfected cells were used as nucleus donors in SCNT and their development rates to the blastocyst stage were estimated. In the present study, it was found that the histone acetylation (H3K9/K14) levels were generally higher in treated groups compared with nontreated control groups. Regarding blastocyst development rate, the OKSM/5-aza-dC (10/49, 20.1 ± 5.9%) combination was better than the other groups: OKSM/TSA (8/77, 11.1 ± 6.9%), OKSM/2i (8/84, 9.5 ± 1.5%), OKSM (6/50, 12.6 ± 6.5%), and negative control group (9/94, 9.6 ± 3.8%; P < 0.05). Embryo development rates appeared to be higher in OKSM/inhibitor-treated groups, although the rate was significantly lower (P < 0.05) in OKSM/2i. In conclusion, our data suggest that transfection with 4 genes and incubating with 5-aza-dC can significantly improve the development rate of bovine SCNT embryos.
53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS
Authors: Olivera R. , Alvarez C. , Stumpo I. , Vichera G.
Published: December 2012

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.
63 QUALITY OF BOVINE EMBRYOS PRODUCED IN VITRO FROM IMMATURE OOCYTES TREATED WITH A SUBLETHAL HYDROSTATIC PRESSURE
Authors: Díez C. , Trigal B. , Caamaño J. N. , Muñoz M. , Correia E. , Martín D. , Carrocera S. , Pribenszky C. , Gómez E.
Published: December 2012

High hydrostatic pressure (HHP) treatment of immature porcine oocytes improves embryo development rates and cell numbers (Pribenszky et al. 2008 Anim. Reprod. Sci. 106, 200–207). However, it is unknown if similar effects can be obtained with bovine oocytes and how HHP affects cryopreservation of the developed blastocysts. In this work, we analyzed the effect of an HHP treatment (Cryo-Innovation Ltd., Budapest, Hungary) on bovine cumulus–oocyte complex (COC) as determined by their developmental ability and embryo quality. Immature COC were submitted to a pressure treatment (200 bar, 1 h at 37°C; HHP group; n = 643) in HEPES-buffered TCM199. Simultaneously, a group of COC was held at 37°C for 1 h (T group; n = 304) in HEPES-buffered TCM199, while other COC were untreated (n = 1182). After in vitro maturation, COC were fertilized in vitro (IVF) and cultured in modified SOF + 6 g L–1 BSA (Holm et al. 1999 Theriogenology 52, 683–700), and embryo development was recorded (5 replicates). Day 7 and 8 excellent- and good-quality embryos were selected for vitrification (cryologic vitrification method; Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). After warming, vitrified blastocysts were cultured in modified SOF + 6 g L–1 BSA + 10% FCS for 48 h (3 replicates). Those blastocysts hatching after warming (at 24 and 48 h) were fixed and stained for differential cell counts. Data were analyzed by ANOVA and REGWQ test and are presented as least squares means ± standard error. The HHP-treated oocytes showed increased development rates on Day 3 (Day 3 ≥5-cell embryos: 64.5 ± 2.9a, 53.4 ± 3.9b, 56.7 ± 2.2b for HHP, T, and untreated groups, respectively; a v. b: P < 0.05); however, D8 blastocyst rates were not affected by the pressure treatment (28.5 ± 1.6, 26.4 ± 2.2, and 27.8 ± 1.3 for HHP, T, and untreated groups, respectively). Treatment did not affect survival rates to vitrification (2-h re-expansion rates: 100 ± 6.7, 100 ± 6.7, and 95.4 ± 6.7; 48-h hatching rates: 58.1 ± 9.4, 71.2 ± 9.4, and 62.3 ± 9.4, for HHP, T, and untreated, respectively). Embryos that hatched after warming did not differ in inner cell mass and trophectoderm cell counts (inner cell mass: 15.0 ± 1.9, 12.7 ± 3.0, and 13.0 ± 2.0; trophectoderm: 133.6 ± 8.4, 137.3 ± 12.8, and 138.4 ± 8.6 for HHP, T, and untreated groups, respectively; P > 0.05). Complementary studies are needed to analyze the effects of a sublethal stress in bovine oocytes on the subsequent embryo production and quality. Species-specific mechanisms could underlie the differences in results obtained in bovine and porcine.
RTA2011-00090 (FEDER-INIA). Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.