인체 트롬보포이에틴(hTPO)은 megakaryopoiesis 과정에 주요한 역할을 하는 사이토카인이다. 따라서 이러한 트롬보포이에틴을 유선조직에서 직접적으로 발현시키기 위하여 소 베타 카제인 프로모터, 인체 트롬보포이에틴 cDNA 및 네오유전자로 구성된 발현벡터를 구축하였다. 소 귀조직 세포로부터 유도된 섬유아세포에 lipoffctamine을 이용하여 발현벡터(pBT-L n대)의 삽입을 유도하였다. G4l8 저항성을 지닌 세포의 콜로니 형성을 유도하기 위하여 2주 이상 배양을 실시하였다. 형질전환 콜로니는 PCR에 의해 동정하였으며, 이들 콜로니를 핵치환 전까지 계속적으로 증식을 유도하였다. 형질전환 세포에 의해 재구성된 난자는 전기적인 융합과 calcium ionophore와 6-DMAP를 이용한 활성화를 실시하였으며, 체외에서 7일간 배양을 실시하였다. 총 35개의 콜로니를 PCR에 의해 분석한 결과, 이 중 29(82.9%)개가 형질전환된 콜로니였다. 형질전환된 세포로 재구성된 난자의 난할율 및 배반포로의 발달율은 65.1%와 23.8%로 나타났다. 형질전환된 세포로 재구성된 난자로부터 발달한 29개의 배반포 중 27개가 형질전환으로 확인되었다. 따라서 이러한 결과들은 형질전환 소 수정란을 형질전환된 세포를 이용한 체세포 복제 기법을 통해 효과적으로 생산할 수 있다는 것을 제시하고있다.

Among the developed bío-degradable polymer fílms as compared wíth transparent fílm(Whíte), black polymer fílm was sígnífícantly more effectíve for controllíng weeds ín red pepper. Also, we found that whíte and black polymer mulchíng had 81.8% and 97.9% of managing weed controllíng effects in rice, respectively. Compared to nonmulched rice paddy with water supply, the non-mulched rice paddy wíthout any water supply has stopped its growth at 41 days after transplantíng, whíle polymer -mulched rice paddy without water supply had about 60% of normally growing rice plants. This shows the polymer treatment has a remarkable effect on water and power saving, solution of herbicidal resistance, avoidance of herbícidal influence to eco-system etc. When the naturally decomposíng polymer was used, a temperature was elevated as high as 4.7 ℃ on maximum and 2.6 ℃ on average. Also the naturally decomposing polymer accelerated rooting by 7 days and lowered a stress level from transplanting. The weed control effect mulched by polymer was remarkable as 98. 7%. The p이ymer now, after 294 days treated on the rice paddy, has been completely decomposed.

The development of humanized culture system of human embryonic stem cells (hESCs) hold promise for therapeutic applications. However, conventional culture system contain animal-derived components such as fetal bovine serum and mouse embryonic fibroblasts that bear a risk of transmitting non-human pathogens and incorporation of non-human immunogenic molecules to hESCs. In this study, we developed an efficient xeno-free hESCs culture system using humanized materials, the CELLstartTM, human foreskin feeder and xeno-free medium containing knockOutTM SR XenoFree (XF-medium) without animal-derived material. The hESCs were gradually adapted to the XF-medium; 25:75, 50:50, 75:25 and 100:0. Two karyotypically normal hESC lines, SNUhES4 and H1, were used for the experiments of xeno-free culture condition. The attachment rates at xeno-free culture system were 52.6±12.4%, 67.0±16.6%, 59.0±13.9%, 28.3±2.9% in SNUhES4, 79.3±5.4%, 53.8±20.9%, 69.4 ±6.4%, 59.8±12.6% in H1 and the spontaneous differentiation rates were 42.2±12.7%, 31.4±2.9%, 40.8±14.5%, 55.2±35.5% in SNUhES4, 35.6±8.5%, 36.4±13.5%, 48.4±7.8%, 80.1±6.0% in H1 in the first four passage. Although the attachment rates were low and the spontaneous differentiation rates were high compared to that of conventional system in the early passages using this humanized culture condition, hESCs in this culture condition were found to maintain hESC characterizations; morphology, expression of cell surface markers and stable karyotype. Our results indicate that simplified compositions of humanized culture system can be applicable to the further optimization for a xeno-free culture of hESCs without the loss of pluripotency and contamination from xenogenic sources.

Controllable transgenic expression systems in transgenic animal model are valuable to the development of therapeutic approaches in human medical fields. The aim of this study was to 1) produce a transgenic cloned dog using inducible tetracycline vector system, and 2) investigate whether the transgenic cloned dog could be induced the transgene expression using doxycycline (Doxy). Canine fetal fibroblasts were infected with retroviral vectors designed to express the enhanced green fluorescent protein (eGFP) gene under the control of tetracycline-inducible promoter. For somatic cell nuclear transfer (SCNT), nucleus of an in vivo matured oocyte was removed and an eGFP expressed cell cultured with 1 ㎍/㎖ of Doxy was injected. After electrical fusion and chemical activation, the reconstructed embryos were transferred to a recipient and pregnancy diagnosis was performed by ultrasonography. Experiment I evaluated the mean fluorescence intensity (MFI) of infected cells while the cells were cultured in the presence of 1 ㎍/㎖ of Doxy for 5 days, and then in the absence of Doxy for 7 days using fluorescence-activated cell sorter. Experiment II was designed to produce an eGFP controllable transgenic cloned dog via SCNT. For verification of transgenic dog, experiment III was performed Southern Blot analysis and observation in vivo regulation of eGFP expression in the cloned dog treated with 100 ㎎/㎏ of Doxy every 2 days for 2 weeks under ultraviolet light. In experiment IV, western blot was used to detect eGFP increase and decrease in skin tissues of transgenic dog under the presence or absence of Doxy. In the results of experiment I, the MFI for infected cells was rapidly increased to approximately 42.3 times after 3 day-treatment compared to pre-treatment and quickly decreased 3 days after ceasing the treatment. In experiment II, a total of 203 embryos were transferred to nine recipients and three pregnant delivered three pups (Tet-on eGFP 0, Tet-on eGFP 1, and Tet-on eGFP 2) by C-sec and Tet-on eGFP 2 among them is still alive. All cloned pups were genetically identical to the donor cell. Tet-on eGFP 2 showed an apparent in vivo eGFP expression on her body after Doxy administration in experiment III. The result of Sothern blotting showed that the transgene insertion was detected from the three cloned dogs and all organs of Tet-on eGFP 1. Experiment IV indicated that a robust eGFP expression in skin tissue of Tet-on eGFP 2 rapidly increased after Doxy treatment and gradually decreased to basal level on 9 weeks after ceasing the treatment. In conclusion, we report here for the first time an inducible transgenic system in canine species and it can stably induce the transgene expression at intended time. This study has demonstrated the capacity to generate transgenic model dog which could regulate the transgene and it would contribute to human medical research fields.

The cloning of canids was succeeded in 2005, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay of successful somatic cell nuclear transfer (SCNT)was due to the unique reproductive characteristics of the female dogin comparison to other domestic mammals, such as ovulation of immature canine oocyte and a requirement of 25 days for the completion of meiosis within the oviduct (Holst & Phemister, 1971). When the technology for the recovery of in vivo matured oocyte was established, the application of cloning also became possible and cloned dog offspring were obtained. This report summarizes the progress of technical procedures that are required for cloning canids and the application of this technique. The first cloned dog, Snuppy, was achieved using an in vivo-matured oocyte which was enucleated and transferred with an adult skin cell of male Afghan hound. After establishment of a criterion of well-matured oocyte for the improvement of SCNT efficiency, we obtained three cloned female Afghan hound and a toy poodle cloned from 14 year-old aged Poodle using SCNT through this factor. To date, cloned dogs appeared to be normal and those that have reached puberty have been confirmed to be fertile. Through application of canine SCNT technique, first, we demonstrated that SNCT is useful for conserving the breed of endangered animal from extinction through cloning of endangered gray wolves using inter-species SCNT and keeping the pure pedigree through the cloning of Sapsaree, a Korean natural monument. Secondly, we showed possibility of human disease model cloned dog and transgenic cloned dog production through cloning of red fluorescent protein expressing dog. Finally, SCNT can be used for the propagation of valuable genotypes for making elite seed stock and pet dog. In summary, dog cloning is a reproducible technique that offers the opportunity to preserve valuable genetics and a potential step towards the production of gene targeted transgenic cloned dogs for the study of human diseases.