The six kinds of natural factors(It refers to wind, cold, summer-heat, dampness, dryness and fire.) is constantly moving to maintain the balance of whole climate of one year. Four seasons are the largest mediation of whole climate of one year. The cold and the heat have the relationship of mutual intervention. The dampness and the dryness have it also. In this process they raise various climate appearance. And that various climate appearance influence the whole lives on the earth. This paper is concerned with the classification of them.

Five elements motion and six kinds of natural factors influence not only human disease but also difference of medicine effect. Because the climate of summer is very hot, that is easy to increase the properties of hot medicines, a doctor must be careful to use it in summer. Also the climate of winter is very cold, that is easy to increase the properties of cold medicines, a doctor must be careful to use it in winter. But in the certain conditions to use diaphoretics or purgatives, in spite of that regulation a doctor can use them.

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.

The efficiency of transgenic livestock production could be improved by early screening of transgene-integration and sexing of embryos at preimplantational stages before trasferring them into recipients. We examined the effciency of multiplex PCR analysis for the simultaneous confirmation of the trasgene and sex during the preimplantational development of bovine embryos and the possibility of green fluorescent protein(GFP) gene as a non-invasive marker for the early screening of transgenic embryos. The GFP gene was microinjected into the male pronuclei of bovine zygotes produced in vitro. The injected zygotes were co-cultured in TCM-199 containing 10% FCS with boving oviductal epithelial cells in a 5% CO2 incubator. Seventeen(13.0%) out of 136 gene-injected bovine zygotes developed by multiplex PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Eight(67%) of 12 embryos at 2-cell to blastocyst stage were positive in the PCR analysis, but only two(11.8%) of 17 blastocysts expressed the GFP gene. Their sex was determined as 7 female and 5 male embryos by the PCR analysis. The results indicate that the screening of GFP gene and sex in bovine embryos by PCR analysis and fluorescence detection could be a promisible method for the preselection of transgenic embryos.

The efficiency of transgenic livestock animal production may be improved by early selection of transgenci preimplantation embryos. To examine the possibility of GFP gene as a non-invasive marker for the early screening of transgenic embryo, the GFP gene was microinjected into rabbit zygotes and the later stages of preimplantation embryos were examined for the expression of GFP. The presence of injected DNA was detected by PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Out of 108 GFP gene-injected rabbit zygotes, seventy three(67.6%) were fluorescence-positive. When 11 fluroresecence-positive blastocysts were analyzed for the presence of GFP gene by PCR, 6(54.5%) were positive, and all of the 8 flrouescence-negative blastocysts were also negative by PCR. The results indicate that the screening of transgene in rabbit embryos by PCR analysis and GFP detection could be a promising method for the preselection of transgenic embryos.

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.