NARO

The 2nd Japan – Korea – China
Joint Symposium

Current and Future Technologies for
Animal Reproduction

September 5, 2012

University Hall
University of Tsukuba
1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 JapanSRD-NILGS 第 2 回 日・韓・中交流シンポジウム

SRD-NILGS the 2nd Japan-Korea-China Joint Symposium

Theme: “Current and Future Technologies for Animal Reproduction”
Location: University of Tsukuba, University Hall
Date and time: September 5 (Wed), 2012; 12:30-18:00

Society for Reproduction and Development (SRD)
http://reproduction.jp/index-j.php
NARO Institute of Livestock and Grassland Science (NILGS)
http://www.naro.affrc.go.jp/org/nilgs/eng/index-e.html

Program
Opening Remarks 12:30-13:00
Chair: Noboru MANABE and Junyou LI
Kei-ichiro MAEDA (President, The Society for Reproduction and Development).
Dong-Il JIN (President, The Korean Society of Animal Reproduction)
Shi-En ZHU (Chairperson, Reproduction Branch of Chinese Association of Animal Science andVeterinary Medicine)
Session 1. Efficient Offspring Production 13:00-14:00
Chair: Hiroko TSUKAMURA and Deshun SHI
JKC-1 Monitoring Metabolic Health of Transition Cows: The Keys to Improve Reproductive
Performance in Dairy Cattle
Toshihiko NAKAO1,2 and Martina HOEDEMAKER1 (1University of Veterinary Medicine
Hannover, Germany; 2Alexander von Humboldt Foundation Alumni Research Fellow fromJapan)P. 6

JKC-2 The Research Progress of Hormone Genetic Immunization Techniques Improving
Growth and Reproduction in Animals
Liguo YANG, Aixin LIANG, Li HAN, Guohua HUA and Shujun ZHANG (Key Laboratoryof Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Collegeof Animal Science and Technology, Huazhong Agricultural University, Wuhan, China)

P. 8

Session 2. In Vitro Embryo Production, Transgenesis and the Related Studies 14:00-16:20
Chair: Nam-Hyung KIM and Eimei SATO
JKC-3 Culture of Bovine Oocytes in an Individually Identifiable Manner and Its Applications
Satoko MATOBA1, 2, #, Trudee FAIR2 and Patrick LONERGAN2 (1Reproductive Biology andTechnology Division, National Livestock Breeding Center, Japan; 2School of Agriculture andFood Science, University College Dublin, Ireland; #Present address, Animal Breeding andReproduction Research Division, NARO Institute of Livestock and Grassland Science, Japan)
P. 10
JKC-4 Production of Transgenic Pigs for the Translational Research
Yubyeol JEON, Seong-Sung KWAK, Seung-A JEONG, Eui Bae JEUNG and Sang-Hwan
HYUN* (*Presenting author; Laboratory of Veterinary Embryology and Biotechnology(VETEMBIO), Collage of Veterinary Medicine, Chungbuk National University, Cheongju,
Korea)

P. 12

Coffee Break 15:00-15:20
Chair: Li-guo YANG and Tae-Woan KIM
JKC-5 Productions of Transgenic Pigs for Xenotransplantation in Korea
Seongsoo HWANG, Keon Bong OH, Dae-Jin KWON, Sun A OCK, Gi-Sun IM, Sung Soo
LEE and Jin-Ki PARK (Animal Biotechnology Division, National Institute of Animal Science,
Rural Development Administration, Korea)

P. 14

JKC-6 Roles of Neurotrophins in Mammalian Reproduction
Xu ZHOU (College of Animal Science and Veterinary Medicine, Jilin University, Changchun,
China)

P. 16

Session 3. Future Possibility of Cloning and Stem Cells 16:20-17:50
Chair: Yuji HIRAO, Gyu-Jin RHO and Xu ZHOUJKC-7 Future Possibility and Current Status of Animal Stem Cell Technology
Hiroshi KAGAMI (Faculty of Agriculture, Shinshu University, Nagano, Japan)

P.18

JKC-8 New Paradigm and Standard for Pluripotent Stem Cells in Domestic Animals
Chang-Kyu LEE, Jin-Kyu PARK, Gwang-Hwan CHOI and Dong-Chan SON (Departmentof Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for
Agriculture and Life Science, Seoul National University, Seoul, Korea)

P. 20

JKC-9 Generation of Buffalo Induced Pluripotent Stem cells
Deshun SHI, Yanfei DENG, Qingyou LIU and Jianrong JIANG (Faculty of Animal Scienceand Technology, Guangxi University, Nanning, China)

P. 22

Closing Remarks 17:50-18:00
Chair: Kazuhiro KIKUCHI
Takashi NAGAI (Chair, The 105th SRD Annual Meeting).

ABSTRACTS

Session 1. Efficient offspring production

JKC-1

Monitoring Metabolic Health of Transition Cows: The Keys to
Improve Reproductive Performance in Dairy Cattle
Toshihiko NAKAO1,2 and Martina HOEDEMAKER1
1
2

University of Veterinary Medicine Hannover, Germany
Alexander von Humboldt Foundation Alumni Research Fellow from Japan

Decrease in fertility of dairy cows has been reported worldwide for the last few decades. The
underlying cause of the declining fertility is metabolic challenge facing cows during transition
periods and concomitant occurrence of peri-parturient diseases. Cows with sever degree of metabolic
challenge have reduced immune and reproductive function, leading to retention of fetal membranes,
metritis, poor quality of the oocyte, and abnormal resumption of ovarian cycles postpartum. If theoocyte is damaged, any conventional hormone treatment or intra-uterine treatment could not be
useful to improve fertility. The most prominent metabolic change in transition periods is theelevation of serum NEFA as a consequence of negative energy balance. High serum NEFA 1 to 2 w
pre-partum is associated with increased incidence of peri-parturient diseases which are major causesof the decreased fertility. Cows with high serum NEFA pre- and/or postpartum showed a higher
incidence of subclinical ketosis, delayed first ovulation, a lower pregnancy rate and a longer intervalfrom calving to conception than those with the normal NEFA concentrations. Subclinical ketosis
with high BHBA in early lactation was also associated with increased risk of metritis, displacementof abomasums, poor fertility, and decreased milk production. The changes in metabolism andimmune function precede common reproductive diseases by a few or several weeks. It is, therefore,
imperative to monitor metabolic changes of cows in transition period and to implement nutritionaland management programs to minimize the negative energy balance in order to improve reproductive
performance. Based on the energy status of cows monitored by serum NEFA, the following
management program can be utilized; (1) high pre-partum NEFA- searching the causes of low dry
matter intake (DMI) and increasing DMI, (2) high NEFA pre- and postpartum, or low NEFA
pre-partum and high NEFA postpartum- increasing DMI, delaying rebreeding, or using embryotransfer instead of AI, (3) low NEFA pre- and postpartum- starting to rebreed shortly after a
voluntary waiting period.
Keywords: dairy cow, transition, negative energy balance, NEFA, fertility

JKC-2The Research Progress of Hormone Genetic Immunization
Techniques Improving Growth and Reproduction in Animals
Liguo YANG, Aixin LIANG, Li HAN, Guohua HUA and Shujun ZHANG
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education,
College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, China

We have successfully obtained two types of hormone DNA vaccines against somatostatin andinhibin for improvement of growth and fertility respectively in animals since the starting of theresearch in 1998. The research process and progress of hormone genetic immunization in 15 years
are reviewed in this paper. Our researches focused on three fields to improve the safety, growth or
fertility promoting effect, and simplicity of the vaccine applications. In order to enhance the immune
effect of the hormone DNA vaccines, some adjuvant such as GM-CSF, CpG and VP22 wasreconstructed into the hormone plasmids. In order to improve the safety of the vaccines, we set up an
asd based host-plasmid balanced lethal system instead of antibiotic resistance genes. Moreover,attenuated Salmonella entericaserovar Typhimurium C500 with crp- asd- double deleted was used asa delivery system to improve the safety and simplicity. So far, we have constructed several decadesof DNA vaccines and compared their results in safety, growth or fertility promoting effect. Our
researches have proved that the better DNA vaccine against somatostatin is C500 (pVGS/2SS-asd)
which caused 31.49% and 25.38% increase in growth of piglets after double immunizations 4 and 8
weeks after weaning compared with the controls. And the effective DNA vaccine against inhibin is
C500 (pVAX-IS-asd) which could improve the average number of litter size (2.5) and mature
follicles (4.9) of immunization group in mice, in comparison to control group. Furthermore, thegenomic DNA was assayed for integrated plasmid using a sensitive PCR method, and the risk ofhormone DNA vaccines due to integration in mice was negligible. Nowadays, assessment of theenvironmental release test of Ministry of Agriculture of genetically modified organisms safety forthese hormone vaccines have been accomplished and the further production test is ongoing.
This study was financially supported by the National Natural Science Foundation of China (No.
30972099).

Session 2. In vitro embryo production, transgenesis and the related studies

JKC-3

Culture of Bovine Oocytes in an Individually Identifiable
Manner and Its Applications
Satoko MATOBA1, 2, #, Trudee FAIR2 and Patrick LONERGAN2
1

Reproductive Biology and Technology Division, National Livestock Breeding Center, Japan
School of Agriculture and Food Science, University College Dublin, Ireland#
Present address, Animal Breeding and Reproduction Research Division, NARO Institute of Livestock andGrassland Science, Japan
2

Although cattle embryos usually develop alone in vivo, in vitro culture of single oocytes orembryos results in poor embryo development. To compensate for this limitation, in vitro culture ofoocytes and zygotes generally takes place in groups to allow the autocrine and/or paracrine factors to
support embryo development replacing the appropriate environment in vivo provided by theirmother’s reproductive tract. However, traditional embryo culture in groups makes tracking ofindividual embryo impossible. Furthermore, although, good quality immature oocytes are selectedbased on their morphology for in vitro maturation (IVM), fertilisation (IVF) and culture (IVC), thismorphological criterion is inadequate; only 30 % of cultured oocytes develop to the blastocyst stage(Day 7), the reminder undergoing developmental arrest prior stage.
Recently, much effort had been made to develop embryo culture systems which facilitate both theidentification/tracking of individual embryos and the supporting effect of autocrine and/or paracrine
factors. Establishment of individual culture systems for oocytes and embryos would greatly facilitate
the study of oocyte developmental competence including the assessment of the relationship between
the follicle and oocyte. Previously, most reports of individual culture procedures have mainly
focused on the post-insemination development of fertilised oocytes. A few studies have attempted
the culture of single immature oocytes; however, these attempts were characterised by low blastocyst
formation. Thus, it is necessary to improve individual culture from the immature oocyte to theblastocyst stage. In particular, it is important to overcome the compromised developmental ability of
individually cultured oocytes.
We examined three systems that have been reported for the individual culture of mammalianembryos; 1) the well of the well, or WOW system, 2) culture on an adhesive matrix, such as Cell-Tak(a formulation of polyphenolic proteins extracted from marine mussel Mytilus edulis) and 3) culture
within the opening of a monofilament polyester mesh. In each of these culture systems, 20 oocytes
are cultured in the same droplet through IVM, IVF and IVC to enhance development. Fixing theposition of oocytes/embryos and the distance between them in the common culture droplet can
provide the double benefit of stimulation by putative autocrine/paracrine growth factors as in group
culture and the individual tracking of oocytes/embryos culture for the entire period of in vitro
development.
We investigated the value of intrafollicular parameters for the non-invasive prediction ofdevelopmental potential of bovine oocytes (i.e. to development to the blastocyst stage) in vitro using
culture methods which permit the individual tracking of oocytes and embryos. To indentify
follicle-related makers of oocyte developmental competence, we investigated the relationshipbetween the levels of several candidate factors including intrafollicular steroids, metabolomic
profiles (amino acid, urea, glucose, citric acid, carbohydrate and fatty acid) in follicle fluid andmRNA abundance of candidate genes in theca, granulosa and cumulus cells and the in vitro
development of oocytes/embryos. The concentration of testosterone, progesterone and estradiol infollicular fluid were not different between oocytes achieving or failing to develop to the blastocyst
stage. L-alanine, glycine, L-glutamate and citric acid concentration were positively correlated,
whereas urea and carbohydrate concentration were negatively correlated with blastocyst formation(P<0.05). Palmitic acid and total saturated fatty acid (SFA) levels were lower, whereas Linolenicacid level was higher in follicle fluid of competent oocytes compared with those of incompetent
oocytes (P<0.05). Relative mRNA abundance of ESR1 and VCAN genes in theca, LHCGR in
granulosa cells and TNFAIP6 in cumulus cells related to oocytes developing to blastocysts was
significantly higher than those related to degenerated oocytes (P<0.05).
In summary, established individual oocytes/embryos culture systems can facilitate studies
examining factors affecting oocyte developmental competence. Several follicular parameters
including metabolite and gene expression in theca, granulosa and cumulus cells can be used as
predictors for oocyte developmental competence.
This research was supported by Science Foundation Ireland (grant number SFI/07/SRC/B1156)
and long term overseas dispatched staff under the NLBC program.

JKC-4

Production of Transgenic Pigs for the Translational Research
Yubyeol JEON, Seong-Sung KWAK, Seung-A JEONG, Eui Bae JEUNG
and Sang-Hwan HYUN**Presenting author
Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Collage of Veterinary Medicine,
Chungbuk National University, Cheongju, Korea

Transgenic pigs have a potential to be a suitable source for xenotransplantation, disease models,disease resistance and bioreactors. Successful production of the cloned animals using by somatic cellnulcear trasnfer (SCNT) was reported in several species including pigs. Among the cloned animals,
pigs are also essential large-animal models in various types of translational research including
diabetes, cardiovascular disease, cystic fibrosis model, influenza resistant model, dermatology,
toxicology and xenotransplantation. In anatomically and physiologically analogous, pigs are moresimilar to humans than mouse, rat, dog and cow. For this reason, a lot of research on the productionof transgenic pigs has been reported for the field of biomedical studies. Transgenic pigs for thetranslational models provide our understanding of the causes and potential treatments of many
human diseases. However, the use of transgenic pigs for the translational research has been very
limited. This may be due to the low efficiency of the relatively process for producing transgenic pigs.
Production technique of transgenic pigs consists of the following process; selection the specific genefor the disease model, design of the vectors and transfection’s skill, SCNT including cytoplasm
quality, donor cell conditions and reprogramming efficiency, and the selection of surrogate forsurgical embryo transfer. Here, we report production of the SCNT-transgenic pigs throughimprovement of SCNT process.
This work was supported by a grant from Next-Generation BioGreen 21 program (#PJ008121012011), Rural Development Administration, Republic of Korea.
Keywords: somatic cell nuclear transfer, transgenic pigs, translational research

JKC-5

Productions of Transgenic Pigs for Xenotransplantation in
Korea
Seongsoo HWANG, Keon Bong OH, Dae-Jin KWON, Sun A OCK, Gi-Sun
IM, Sung Soo LEE and Jin-Ki PARK
Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration,
Korea

A shortage of organs from deceased human donors is a major problem limiting the number oforgan transplants performed each year. In Korea, in February 2012, the Korean Network for Organ
Sharing (KONOS) waiting list for solid organ transplantation approached 29,267 patients.
To overcome this serious problem, pigs are currently the preferred species for organ
xenotransplantation. Pig seems to be the optimal xenograft donor animal because of a similar size as
human organs, many physiological similarities with humans, short reproduction cycles and large
litters and the maintenance is possible at specific (SPF) or designed pathogen free condition (DPF).
More than 10 different types (DAF, CD59, Fas ligand, HA-hHO1, and shTNFR1Fc, etc) oftransgenic cloned pigs for xenotransplantation (xeno-pig) have developed in Korea. Especially 4
types of xeno-pigs such as α1,3-galactosyltransferase knock-out (+/-) (GalT KO +/-), GalT KO
additionally knock-in for the human CD46 (GalT KO/hCD46 KI), human CD73 transgenic, and
GalT KO -/- have generated in National Institute of Animal Science.
Now GalT KO -/- xeno-pigs have become herd by natural mating and SCNT using GalT KO -/cells and their organs such as heart and kidney have applied pig-to-primate xenotransplantation.

JKC-6

Roles of Neurotrophins in Mammalian Reproduction
Xu ZHOU
College of Animal Science and Veterinary Medicine, Jilin University, Changchun, China

Neurotrophins (NTS) are required for the proliferation, differentiation, survival, and death ofneuronal cells. The family of neurotrophins are composed of (nerve growth factor) NGF,
brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4(NT4), threetropomyosin-related kinase (Trk) receptors (TrkA, TrkB and TrkC) and a low affinity receptor
p75NTR. There is evidence to suggest that NTs not only have effects on the nervous system, but also
play important roles in regulating the development of non-neuronal cells such as in reproductive
systems. During the pass years, a series of work has been done in our laboratory to study theexpression and function of NTs in female bovine and porcine reproductive systems, including
oocytes and embryos, and in ejaculated spermatozoa from bull and men. We have shown that GV
oocytes, MII oocytes, 4-cell and 8-cell embryos, morulae, and blastocysts were all shown to express
BDNF. The highest expression was detected in MII oocytes and the lowest expression in 8-cellembryos. The mRNA for BDNF was highly expressed in the PA and IVF blastocysts compared to
the NT blastocysts. BDNF caused a significant increase in the rates of in vitro-fertilized blastocyst
formation and parthenogenetic blastocyst formation. BDNF, NGF, NT4, TrkA and TrkB mRNAs
and proteins were detected granulosa cells, cumulus oocyte complexes, theca cells of follicles, aswell as in the oviduct epithelial cells, endometrium in uterus of cows and sows. BDNF and NGF
promoted the growth of granulosa cells. NGF/TrkA and NT-4/TrkB pathway can simulate theexpression of FSHR and LHR mRNA expression in bovine granulosa cells. LH and FSH canstimulate the mRNA expression of NGF and its receptor TrkA. BDNF, NGF, NT-4, TrkA and TrkB
exist in mature spermatozoa from bull and men. NTs can modulate the function of bovine and humanspermatozoa and may be a biomarker for infertility of men.

Session 3. Future possibility of cloning and stem cells

JKC-7

Future Possibility and Current Status of Animal Stem Cell
Technology
Hiroshi KAGAMI
Faculty of Agriculture, Shinshu University, Nagano, Japan

Recent progress on stem cell technology could lead breakthrough in animal reproduction.
Reproductive technologies; artificial insemination, cloning, stem cell regeneration, gene transfers, invitro fertilization and sex alteration, have been developed in cattle, swine, chicken etc. These
innovations must be refined and performed in the next decades. Especially, stem cell technology
could be one of the most promising subjects in animal reproduction (Sartori et al., 2012). Embryonicstem cells (ES cells) from ICM of mouse blastocysts was isolated (Martin and Evans, 1975). Whenthe cells were injected into recipient embryos, somatic and germline chimeras were generated.
Offspring from donor ES cells derived gamete could be generated. It was found that the stem cells
contained self-renewal ability and maintained pluripotentcy both in vivo and in vitro. Successful
application of these stem cell technologies could improve genetic performance of livestock
effectively. Induced Pluripotent Stem cells (iPS cells) was developed by retrovirus-mediatedtransfection with four transcription factors; Oct-3/4, Sox 2, KLF 4 and c-Myc, into mouse fibroblasts(Takahashi and Yamanaka, 2006). The stem cell technology involved ES or iPS cells could
expenditure to human health. A large number of drugs, diagnostic probes and vaccines are frequently
applied to livestock prior to becoming available for use by humans. Many of the pharmaceuticalindustry have had much interests for livestock production since the research findings could bedirectly applicable to human medicine. One of the most promising applications of the stem cell
technology should be regenerative medicine. The success could bring novel strategies for the medical
treatment.
Thus, the stem cells should be one of the most useful tools (Kagami et al., 2006) for future
production of transgenic animals, generation of pharmaceutical substrates, sexual manipulation ofoffspring, conservation of endangered animals and generation of resistant animal for infectious
disease. Moreover, genetic modification into the stem cell will enhance improvement of livestock
breeding. Accumulation of the scientific knowledge and technical innovations should open up new
frontier for animal reproduction and frontier medicine (Robinton and Daley, 2012).
References:Kagami H et al. (2006) Pluripotent cell culture engineering and the application for biotechnology. In: Animal
Cell Technology, Basic and Applied Aspects, Springer, 135-141.
Martin GR and Evans MJ. (1975) Differentiation of clonal lines of teratocarcinoma cells: formation ofembryoid bodies in vitro. Proceedings of the National Academy of Science USA, 72, 1441–1445.
Robinton DA and Daley GQ. (2012) The promise of induced pluripotent stem cells in research and therapy.
Nature, 481, 295-305.
Sartori C et al. (2012) Ovine-induced pluripotent stem cells can contribute to chimeric lambs. Cellular
Reprogramming, 14, 8-19.
Takahashi K and Yamanaka S. (2006) Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors. Cell, 126, 663-673.

JKC-8

New Paradigm and Standard for Pluripotent Stem Cells in
Domestic Animals
Chang-Kyu LEE, Jin-Kyu PARK, Gwang-Hwan CHOI and Dong-Chan SON
Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for
Agriculture and Life Science, Seoul National University, Seoul, Korea

Since pluripotent cells were first derived from inner cell mass (ICM) of blastocyst in mouse,many effort have been made to establish pluripotent stem cells from domestic mammals. However
for many domestic species including the pig it remains that authentic pluripotent stem cells fromembryos of domestic animals have not yet been derived. It has proven difficult to maintainembryonic stem cells (ESCs)-like state in domestic animals due to the frequent occurrence of
spontaneous differentiation into epiblast stem cells (EpiSCs)-like state during culture based on
genomic background. A recent study has reported that pluripotent stem cells exist in one of two
forms and can be categorized according to their pluripotent state. The first is a “naïve” state which ischaracterized by small, round or dome-shaped colony morphologies and LIF signaling, mouse ESCs
(mESCs) represent this ES cells of this type. A second “primed” state has also been described within
which EpiSCs or human ESCs (hESCs) can be placed. These primed state pluripotent stem cells
display flattened monolayer colony morphologies and FGF/ Nodal/Activin signaling pathways. We
suggest that pluripotent stem cells derived from embryos and iPSCs derived from embryonic
fibroblasts in the domestic model, especially in pig, possess a primed pluripotent state similar to thatof mEpiSCs or hESCs, rather than to that of mESCs. Meanwhile, A few studies have reported that
non-permissive species could be derived to a naïve pluripotent state using various exogenous factors
including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions and up-regulation of Oct3 or
klf4. In present, we are trying to establish naive pluripotent stem cells with 2i and other factors andwe have been able to successfully induce PEFs into a LIF- dependent naïve pluripotent-like cell line
showing a mESC-like morphology. Further characterization of these cells is in progress.
This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development
Administration, Republic of Korea.

JKC-9Generation of Buffalo Induced Pluripotent Stem cells
Deshun SHI, Yanfei DENG, Qingyou LIU and Jianrong JIANG
Faculty of Animal Science and Technology, Guangxi University, Nanning, China

Ectopically expression of defined factors could reprogram mammalian somatic cells into induced
pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines.
Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells
or inner cell mass, but the efficiency is extremely low. Here, we report a successful method toreprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells (biPSCs) by transduction of
buffalo defined factors (Oct4, Sox2, Klf4 and c-Myc) using retroviral vectors. The established
biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype,positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2,
Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3 and FOXD3. They could form
embryoid bodies in vitro and teratomas after injecting into the nude BALB/C mice, and three germlayers were identified in the embryoid bodies and teratomas. Methylation assay revealed that, thepromoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs,
while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs.
Furthermore, inhibiting p53 expression by co-expression of SV40 large T antigen and buffalo
defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase theefficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with
biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into
biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased byinhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo
stem cell signal pathways, establishing buffalo stem cell lines and producing genetic modification
buffaloes in the future本シンポジウムは、社団法人つくば観光コンベンション協会からの協力を得て開催されます。

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