Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus

In our previous studies, the cardiac xenotransplantation from an alpha-1,3-galactosyltransferase knockout pig (GT-MCP-MCP) to cynomolgus monkeys showed a mean survival of 38 days. The objective of this study is to genetically upgrade the GT-MCP-MCP pig, to further enhance membrane cofactor protein (MCP) expression and to express an endothelial specific thrombomodulin (TBM). MCP is a complement regulatory protein and TBM is a coagulation inhibitor. As the dicistronic cassette for wild-type-based MCP and TBM concurrent expressions does not show any increase of MCP, we optimized the MCP codon usage (mMCP) and substituted mMCP for MCP. When the mMCP-TBM cassette was transfected to HeLa cells, we were able to find an increased expression of MCP and endothelial cell-specific TBM expression. The cassette was then transfected into ear-skin fibroblasts isolated from one-month-old #23-4 of a GT-MCP-MCP pig, and the cell populations expressing MCP were obtained by MACS cell sorting. We performed a single cell culture of the selected cells, and obtained clones over expressing 90% MCP. The cells of a clone were used as a donor for nuclear transfer and generated GT-MCP/-MCP/mMCP/TBM pig. The transgenic pig was confirmed to be carrying the cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Thus, we believe that the GT-MCP/-MCP/mMCP/TBM transgenic pig would be potential for the prolongation of xenograft survival in the recipients.

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig (GalT-MCP/-MCP). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain GalT-MCP/-MCP/CD39/CD73 pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from GalT-MCP/-MCP pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate GalT-MCP/-MCP/CD39/CD73 pig expressing CD39 and CD73 at endothelial cells.

Tissue engineering (TE) has been developed to create functional organs and tissue by combining 3D matrix and cells in vitro. Vascularization and angiogenesis are utmost important for supply of nutrients and oxygen in tissue engineered organs. The present study was performed to isolate and characterize primary endothelial cells (EC) from aorta of alpha 1, 3-enzyme galactosyltransferase knock out (GalT KO) pig, to minimize immune rejection and analyze body immune system for future xenotransplantation studies. Isolation of primary EC from aorta were performed by incubation with dispase for 8-10 min at 37°C. Primary EC were cultured in EC growth medium on different extra cellular matrix (ECM), either collagen or gelation. Primary EC exhibits morphological characteristics and showed positive expressions of EC specific marker proteins i.e. PECAM1, KDR and VWF despite of their ECM surface; however, on collagen based surface they showed increase in mRNA level analyzed by qPCR. Primary EC cultured on collagen were sorted by flow cytometer using KDR marker and cultured as KDR positive cells and KDR negative cells, respectively. KDR positive cells showed dramatically increased in PECAM1 and VWF level as compared to KDR negative cells. Based on the above results, primary EC derived from GalT KO are successfully isolated and survived continuously in culture without becoming overgrown by fibroblast. Therefore, they can be utilize for xeno organ transfer, tissue engineering, and immune rejection study in future.

돼지의 장기를 영장류에 이식할 때 단시간 내에 발생하는 초급성 면역거부반응 문제를 해결하기 위해 이를 제어할 수 있는 alpha1,3-galactosyltransferase knock out (Gal-T-/-) 돼지를 생산하였다. 그럼에도 불구하고 그 심장을 이식을 받은 영장류가 사망하는 것으로 보고되었으며, 그 원인은 non-Gal 항원-항체 반응에 의한 면역반응과 돼지와 영장류 간 분자, 생리적 차이에 의해 발생하는 급성 체액성 거부반응 때문이라고 알려져 있다. 본 연구는 어떤 인자와 기전에 의해 이식된 장기가 손상되고 사망하게 되는지 분자 수준에 서 알아보기 위하여, 영장류에 이식한 Gal-T-/- 돼지 심장에서 유전자의 발현 변화를 분 석하였다. 이를 위해 동일 부모에서 태어난 Gal-T-/- 돼지의 이식에 활용하지 않은 심장 을 대조군으로 하여 cynomolous 원숭이에 이식 후 9일째 채취한 심장으로부터 총 RNA 를 추출한 후 Sequencing을 통해 전 돼지 유전자의 발현수준을 비교하였다. 분석 결과, 이식된 심장에서 1292개의 유전자 발현이 유의적으로 증가하였고, 949개는 유의적으로 감 소하는 것으로 분석되었다. 이 중에서 심근 경색 등과 같이 심장이 손상되면 발현이 증가 하는 matricellular 단백질 유전자인 tenascin C(23.7배), Tsp-1(13.9배), -2(5.8배), -4(6.6 배), SPARC(3.6)의 발현이 증가한 것으로 분석되었다. 특히 혈관에서 혈액 응고를 촉진 시킨다고 알려진 Tsp-1의 발현이 유의하게 증가한다는 것을 quantitative RT-PCR 분석 으로 확인하였다. 또한 혈액응고의 중요한 조절 인자인 TF의 발현이 증가한 반면 억제 인자인 TFPI와 TBM의 발현은 변화가 없다는 것을 확인하였다. 이러한 결과는 이식 과 정 중에 가해진 생리적 또는 물리적 손상 및 원숭이 혈액의 재관류 자극에 의해 심장의 기능 마커 유전자가 지속적으로 발현되는 것으로 예측된다.

Polydnavirus (PDV) is a group of double-stranded DNA insect viruses. PDV is mutualistic with some ichneumonid and braconid wasps to parasitize specific lepidopteran hosts. The viral genome is located on the wasp chromosome(s) as a proviral form and replicates only at the female reproductive organ during late pupal stage. The viral particles are accumulated in the oviduct lumen and delivered to the parasitized host along with wasp eggs during parasitization. The viral particles enter target tissues in the parasitized larvae and alter host physiological processes for the wasp development by suppressing immune responses and extending larval period. Cotesia plutellae bracovirus (CpBV) is a PDV symbiotic to C. plutellae parasitizing young larvae of the diamondback moth, Plutella xylostella. The viral particles of CpBV encode 157 open reading frames classified into different gene families. CpBV-PTP family is the largest and comprises of at least 40 gene members. CpBV-BEN, CpBV-ELP, and CpBV-IkB families also share common motifs in each gene group. In addition, two homologous genes of CpBV15α and CpBV15β are encoded in a viral genome segment. To apply these viral genes to enhance an alpha-baculovirus in insecticidal activity, they were recombined with AcNPV under a PDV promoter. As a control, under the same promoter, the recombinant baculovirus expressed an enhanced green fluorescence protein (EGFP). Upon injection or oral feeding tests, three different recombinant baculoviruses (AcNPV-ELP1, AcNPV-CpBV15α, AcNPPV-CpBV15β) enhanced the insecticidal activity compared to a control recombinant (AcNPV-EGFP). However, there was a variation in the up-regulation of the insecticidal activities among the recombinants. AcNPV-ELP1 showed the greatest potency in the insecticidal activity against the beet armyworm, Spodoptera exigua, larvae. AcNPV-ELP1 exhibited a significant variation in insecticidal activity among different larval stages of S. exigua. In the fifth instar, 1.435x107 PIB treatment of AcNPV-ELP1 showed a median lethal time at 112.7 h. ELP1 protein was detected in the hemolymph at 24 h after the viral treatment. Foliar spray of AcNPV-ELP1 was performed in pot assay and resulted in 88% control efficacy against S. exigua, while control efficacies of AcNPV-EGFP and bifenthrin (a pyrethroid insecticide) resulted in 65% and 96%, respectively. These results suggest that a PDV gene, ELP1, may be applied to develop a novel control agent by ameliorating commercial microbial insecticides or by generating transgenic crops.

To avoid hyperacute rejection of xenografts, α1,3-galactosyltransferase knock-out (GalT KO) pigs have been produced. In this study, we examined whether Sia-containing glycoconjugates are important as an immunogenic non-Gal epitope in the pig liver with disruption of α1,3-galactosyltransferase gene. The target cells were then used as donor cells for somatic cell nuclear transfer (scNT). A total of 1,800 scNT embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. Real-time RT-PCR and glycosyltransferase activity showed that α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST) in the heterozygote GalT KO liver have higher expression levels and activities compared to controls, respectively. According to lectin blotting, sialic acidcontaining glycoconjugate epitopes were also increased due to the decreasing of α-Gal in heterozygote GalT KO liver, whereas GalNAc-containing glycoconjugate epitopes were decreased in heterozygote GalT KO liver compare to the control. Furthermore, the heterozygote GalT KO liver showed a higher Neu5Gc content than control. Taken together, these finding suggested that the deficiency of GalT gene in pigs resulted in increased production of Neu5Gc-bounded epitopes (H-D antigen) due to increase of α2,6-sialyltransferase. Thus, this finding suggested that the deletion of CMAH gene to the GalT KO background is expected to further prolong xenograft survival.

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of α-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% CO2 in air at 38.5℃ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-α/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.

Preharvest sprouting (PHS) not only causes reduction of grain yield, but also affects the quality of grains, resulting into significant economic losses. PHS is governed by multiple genes. Little is known about the large genetic variation of preharvest sprouting in rice. In the present study, genetic variations of four PHS genes, OsVP1, Osaba1, Alpha-amylase3D and OsGA20ox1 were studied by using whole-genome resequencing data of 84 accessions of rice core set. A total of haplotype groups; 27, 29, 6 and 14, for OsVP1, Osaba1, Alpha-amylase3D and OsGA20ox1, respectively, were detected among the 84 accessions. Some new SNPs and InDels were found in exon part of PHS related genes were expected to result in amino acid changes following analysis of the genetic code variations, and the germplasm or varieties which are resistant to preharvest sprouting were explored. Based on this step, phenotyping for PHS is ongoing, and the association mapping of PHS will be conducted by using SNPs resulted from the haplotyping data. The present results will be ultimately useful to the molecular breeding for the development of PHS resistant rice cultivars.