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.

Variance of conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants, essential for the maintenance of early pregnancy. Similar to other IFN genes such as IFNA and IFNB, multiple IFNT genes are present. However, some kinds of IFNT genes actively transcribed and regulated in bovine conceptuses have not been well characterized. In this study, during the course of bovine IFNT gene transcription through the use of next generation sequencer SOLiD3, revealed that among 38 IFN genes registered, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses during early pregnancy. Also, to identify a transcription factor(s) involved in the regulation of IFNT genes, mRNAs for various known transcription factors were investigated by real-time PCR in conceptus tissues, respectively. Furthermore, compared to the IFNT genes, IFNT1 and IFNTc1 had same active levels, which were previously shown to correlate with the appearance of effective antiviral activity. However, the expression levels of these Luc activities differed. Bovine ear fibroblast (EF) cells were cotransfected with luciferase reporter constructs carrying upstream (–631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1(JUN), ETS2 and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP). CDX2, either alone with the other 2 transcription factors, was found to increase luciferase activity approximately 14- and 11-folds, respectively. The degree of transcriptional activation of the IFNTc1 gene was not similar to that IFNT1 gene by AP1, ETS2 or/and CREBBP, expression plasmid. These results suggest that two isoforms of bovine conceptus IFNT genes are regulated differently in conceptuses during early pregnancy.

Stored grain pests can cause reduction of grain quantity, quality, commercial value and germination rate. Susceptibility of three fumigants, methyl bromide, ethyl formate and phosphine, were assessed on Tribolium castaneum, which is an important stored grain pest. On susceptible insects, LCT50 of phosphine was 0.654mg h/L for egg, 0.127mg h/L for late larvae, 0.105mg h/L for pupae and 0.048mg h/L for adult stage, respectively. LCT50 of methyl bromide was 33.193mg h/L for egg, 14.585mg h/L for late larvae, 8.616mg h/L for pupae and 11.967mg h/L for adult stage, respectively. LCT50 of ethyl formate were 25.165mg h/L for egg, 80.912mg h/L for late larvae, 176.326mg h/L for pupae and 68.578mg h/L for adult stage, respectively. On resistant insects, LCT50 of phosphine were 82.325mg h/L for egg, 33.315mg h/L for late larvae, 73.546mg h/L for pupae and 55.707mg h/L for adult stage, respectively. LCT50 of methyl bromide were 19.250mg h/L for egg, 43.413mg h/L for late larvae, 76.842mg h/L for pupae and 19.387mg h/L for adult stage, respectively. LCT50 of ethyl formate were 87.552mg h/L for egg, 113.457mg h/L for late larvae, 200.122mg h/L for pupae and 85.394mg h/L for adult stage, respectively.

소나무재선충(Pine wood nematode, PWN)은 아시아와 유럽의 소나무류들에 침입하여 고사시키는 심각해 병원성 선충이다. 가장 완벽한 방제 방법은 감염목을 소각/분쇄의 방법으로 PWN 감염목을 제거하는 것이다. 현재 PWN의 다른 종과 차이를 가지는 유전자서열을 이용한 진단 방법이 다양하게 개발되어져서 실험실에서의 종 판명은 가능한 실정이다. 하지만, 대부분의 분자진단방법은 소나무 목편에서부터의 시료 추출, 그리고 분자진단 시약과 혼합하고, PCR을 이용하여 증폭을 진행 한 후, 결과를 확인하는 과정이 필요하다. 그러므로, 분자진단법을 현장에 적용하기 위해서는 향 후 많은 노력이 필요하다. 이에 반하여 항원/항체 반응을 이용한 진단 방법의 경우는 항원 추출 후, 일정량을 신속진단키트에 떨어 뜨려서 반응을 확인하는 과정을 거치므로, 전문적인 지식이나 기술이 필요하지 않다. 하지만, PWN과 PWN 감염목에 특이적인 항원이 밝혀지지 않은 상황이다. 최근에 우리는 PWN단백질 중에서 분비되는 Aspartic peptidase 1 (ASP1)을 항원으로 선정을 하고, 전장단백질을 Baculovirus Expression System으로 발현을 하였다. PWN-ASP1을 항원으로 단클론항체를 분비하는 세포 주를 확립하였다. 이러한 단클론항체는 향후, PWN의 소나무 침입에 관한 연구와 신속진단키트의 개발에 활용될 수 있을 것이다. (본 연구는 국립산림과학원의 연구비 지원으로 진행되었음)

In order to identify the specific antigens for pine wood nematode (PWN), we confirmed that one of the genes commonly found in the transcriptome, proteome and secretory proteins of PWN belonged to the Aldose Reductase (AR) family protein. 36.5 kDa PWN-AR1 was expressed and purified using Baculovirus Expression System. Total 1,546 hybridoma fusion library was generated and screened for specificity to PWN-AR1 by Enzyme Linked Immunosorbent assay (ELISA). Nine clones showed strong immunoreactivity to PWN-AR1 were limited-diluted. Total 864 limited-diluted clones were further screened using PWN-AR1 by ELISA and 34 monoclonal antibody (Mab) clones were selected. 34 Mab clones were further screened using PWN extracts and a standard PWN-infected pine tree extract by ELISA. Finally nine clones were selected and their immunoreactivities to 4 different nematodes were examined by ELISA. Seven clones pecifically recognized PWN while two clones recognized 4 nematodes. Our data suggested that PWN-AR1 is a PWN secretory enzyme while PWN is invading pine trees, Thus, PWN-AR1-Mabs could be used to develop diagnosis tools for PWN and its infected pine trees. (This work was supported by National Institute of Forest Science)