Naegleria fowleri is pathogenic free-living amoeba leading to primary amoebic meningoencephalitis(PAM) in human and animals. The nfa1 gene cloned from N. fowleri is located on food-cup structure called pseudopodia and function an adherence of host target cells. To evaluate the effect of nfa1 vaccination against N. fowleri infection, we constructed retroviral vector(pQCXIN) expressing nfa1 gene. To determine the effect of vaccination and protective immunity in in vivo models, we measured the immunoglobulin levels, cytokine induction, and survival rate in mice infected with N. fowleri. Both levels of IgG and IgG subclass in DNA vaccinated mice were significantly elevated. The cytokine analysis show that DNA vaccinated mice induces production of IL-4 and IFN-γcytokines suggesting a Th1/Th2 mixed type immune response. The levels of nfa1 specific IgG antibody were maintained highly until 12 weeks post-vaccination in vaccinted mice. The nfa1 vaccinated mice using retroviral vector increased significantly survival rate(60%) after N. fowleri infection. Consequently, the nfa1 vaccination effectively induces protective immunity by upregulation of immune response in mice infected with N. fowleri. These results suggest that DNA vaccination using retroviral vector may be proper trial for treatment and prevention of PAM.

Recently, RNA interference (RNAi) technology has been emerged as a potent tool for pest control strategy. Based on the previous studies on RNAi via leaf disc-mediated systemic delivery of dsRNA and in planta expression of hairpin RNA by agroinfiltration, the coatomer subunit alpha (COPA) gene has been found to be a crucial target for RNAi against Tetranychus urticae. In current study, transgenic plants of Arabidopsis thaliana expressing COPA hairpin RNA were generated by the floral dip method. Putative transgenic plants were screened by PCR and positive transformants were subjected to bioassay using age-synchronized and host-adapted T. urticae. T. urticae feeding on plants expressing dsRNA/siRNA showed more than 80% mortality as compared to the mites feeding on control plants at 6 days post-infestation. Our data shows that in planta expression of hairpin gene such as COPA may serve as an effective way for the control of this important pest in ornamental and economically important plants.

The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.

The principal objective of this study was to clone transgenic embryos in order to improve the efficiency of transgenic animal production by the combination of microinjection and nuclear transplantation techniques. Mature female New Zealand White rabbits were superovulated by eCG and hCG treatments, fllowed by natural mating. Zygotes were collected from the oviducts at 18∼22 h after hCG injection by flushing with D-PBS containing 5% fetal calf serum(FCS). Two to three picoliters of green fluorescent protein(GFP) gene wa microinjected into male pronucleus. The foreign gene-injected zygotes were cultured in TCM-199 or RD medium containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% CO2 incubator. The morulae expressing GFP gene were selected and their blastomeres were separated for the use of nuclear donor. Following nuclear transplantation of fluorescence-positive morula stage blastomeres, 13 (21.3%) out of 61 fused oocytes developed to blastocyst stage and all of the cloned blastocysts expressed GFP. The results indicate that the screening of transgene in rabbit embryos by GFP detection could be a promisible method for the preselection of transgenic embryos. Also the cloning of preselected transgenic embryos by nuclear transplantatin could be efficiently applied to the multiple production of transgenic animals.

Tooth bud is initiated by dental epithelium thickening and invaginated epithelium forms cap-like structure with condensed underlying mesenchyme. From this cap stage, specific epithelial cells form a cluster of non-dividing cells, primary enamel knot (PEK), and this PEK expresses abundant signaling molecules and functions as a signaling center for tooth development. Recently, hundreds of genes involved in tooth development have been reported by advanced genome-wide screening technology, but still remained unidentified genes obstruct elucidation of the precise molecular mechanisms underlying tooth development. In this work, we examined the expression patterns and developmental functions of a novel tooth germ-expressing gene, Dachshund1 (Dach1). Dach1, known as the cell fate regulator via controlling the transcription in various cell types, expressed in PEK region from cap stage to bell stage. In order to evaluate the developmental functions of Dach1 in tooth development, we cultivated the lower first molar at E12.5 for 2 and 4 days with or without treatments of antisense-oligodeoxynucleotides (AS-ODNs) against Dach1. After the knocking down of Dach1, morphological changes and cell physiology such as proliferation, cell death and migration patterns were examined. In addition, the expression levels of PEK-expressing genes such as Bmp2, Bmp7, Fgf4, Shh and Wnt5a were examined by using RT-qPCR and in situ hybridization methods. Based on these results, we suggest that Dach1 would involve in mice tooth morphogenesis through regulating the expressions of PEK related genes and cellular events to form the proper tooth structural formation.

To produce abiotic stress resistant transgenic cucumber, the cotyledonary node explants of cucumber (c.v. Eunsung) were inoculated with A. tumefaciens strain EHA105 containing the binary vector (pPZP211) carrying Nit gene. The 491 explants inoculated with bacterium solution for 30 min were maintained on 50 mg/L paromomycin contained shoot induction (SI) medium for first 2 weeks and then subcultured on 100 mg/L paromomycin to obtain transgenic adventitious shoots for 4 x 14 days. So far, 5 plant were selected, and then acclimated in soil. Of them, 3 transgenic plants with Nit gene were confirmed by Southern blot analysis.

Living modified organisms(LMO) by modern biotechnology has played a highly visible role. But the public concerns relating to the environmental risk of LMO have been raised because of the potential benefits of this new technology. The recently adapted Cartagena Protocol on Biosafety contains an Annex on risk assessment, which specifies in some detail points to consider. All LMO should be evaluated according to their biological properties(phenotypic, genetic, agronomic and ecological characteristics) before release to the environment. To build up the capacity building of risk assessment method for LMO used environmental remediation, we engineered a model plant, Solanum nigrum L. expressing organomercurial lyase gene(merB). MerB transforms methylmercury to less toxic, non-biomagnified ionic mercury. In this report, we compared 8 different phenotypic, agronomic characteristics and rhizosphere microorganisms with control. The difference between transgenic and non-transgenic plant has not found.

Field performance and morphological characterization was conducted on seven transgenic lines of Codonopsis lanceolata expressing γ-TMT gene. The shoots were obtained from leaf explants after co-cultivation with Agrobacterium tume-faciens strain LBA 4404 harboring a binary vector pYBI 121 that carried genes encoding γ-Tocopherol methyltransferase gene (γ-TMT) and a neomycin phosphotransferase II gene (npt II) for kanamycin resistance. The transgenic plants were transferred to a green house for acclimation. Integration of T-DNA into the T0 and T1 generation of transgenic Codonopsis lanceolata genome was confirmed by the polymerase chain reaction and southern blot analysis. The progenies of transgenic plants showed phenotypic differences within the different lines and with relative to control plants. When grown in field, the transgenic plants in general exhibited increased fertility, significant improvement in the shoot weight, root weight, shoot height and rachis length with relation to the control plants. However, all seven independently derived transgenic lines produced normal flower with respect to its shape, size, color and seeds number at its maturity. Indicating that the addition of a selectable marker gene in the plant genome does not effect on seed germination and agronomic performance of transgenic Codonopsis lanceolata. T1 progenies of these plants were obtained and evaluated together with control plant in a field experiment. Overall, the agronomic performance of T1 progenies of transgenic Codonopsis lanceolata showed superior to that of the seed derived non-transgenic plant. In this study, we report on the morphological variation and agronomic performance of transgenic Codonopsis lanceolata developed by Agrobacterium transformation.

This study was carried out to analyze the mineral contents including calcium in brown rice for the rice plants transformed with CAX1 gene. In the previous study the transgenic rice plants overexpressing the Arabidopsis Ca2+/H- antiporter CAX1 gene were de