해상 유통되는 유해액체물질(NLS)의 유출사고대비 물질군 선정을 위해 596종의 NLS를 대상으로 위해성 DB를 구축하고 우선순위 선정시스템을 통해 전체 우선순위를 선정하였다. 우선순위 목록을 바탕으로 2014-2015년 해상유통물질 158종을 추출한 뒤 물질군 구분 기준을 적용하여 0~3순위의 4개 물질군을 제시하였다. 국가차원의 NLS 유출사고대비를 위해서는 물동량 및 유해성이 높은 0~1순위 물질군의 집중관리와 함께 2~3순위 물질군의 정보 구축작업이 지속되어야 한다. 항만별로는 NLS 유통 유형이 다르므로 각 항만의 물질별 유통특성을 파악한 뒤 0~1순위 물질군 위주로 관리하는 것이 효율적일 것으로 판단된다. 또한, 동해남부권역(울산, 부산), 남해중부권역(광양, 여수), 서해중부권역(평택, 대산, 인천)을 NLS 사고대비를 위한 특별관리구역으로 지정하여 해상유통되는 NLS 의 감시·감독을 강화하고 방제 장비, 자재 및 약제를 집중 배치해야 할 것으로 판단된다. 향후, 위해성 DB의 구축과정에서 나타난 위해성 정보 부재 물질들의(만성독성) 지속적인 생산 및 보완이 필요하며, 특히 수생태 독성의 경우 해양생물종을 대상으로 한 자료 생산 및 확보가 지속되어야 할 것이다. 또한, 해상 HNS 사고 관리를 위해서는 HNS 해상유통에 대한 정보를 확인할 수 있는 시스템이 조속히 구축되어야 한다.

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with α1,3-galactosyltransferase gene (α1,3-GT gene), DT-A/pGT5’/neo/pGT3’, DT-A/NLS/pGT5’/neo/pGT3’ and pGT5’/neo/ pGT3’/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5’ recombination arm (pGT5’) and a 1.9-kb fragment as the 3’ recombination arm (pGT3’). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of α1,3-GT locus. DT-A/pGT5’/neo/pGT3’ vector contain only positive-nega-tive selection marker with conventional targeting vector. DT-A/NLS/pGT5’/neo/pGT3’ vector contain positive-negative selection marker and NLS sequences in upstream of 5’ recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5’/neo/pGT3’/NLS vector contain only positive selection marker and NLS sequence in downstream of 3’ recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with 300 μg/ml G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for α1,3-GT gene disruption in 3´ PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5’/neo/pGT3’ knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

This study evaluated the efficiency and compared with different materials of loading vessels for vitrification-plastic/glass, copper grid and nylon. The loading method, vitrification, cryop-reservation and warming method of the oocytes were examined. The loading samples prepared in manual or company-made and sterilized, loaded the COCs selected on each samples and cultured for maturation during 40 hours, and then exposed sequentially to ethylene glycol solution. Thawing method was reversely treated and exposed for warmed oocytes. After oocytes were thawed, fertilized and cultured in vitro for 3-4 hours, rates of development and morphological appearance were examined. The results were as summarized: ㆍOPS from company-made or hand-made of the hematocrit micropipettes, NLS from fishing line and EMG from company-made for EM were used for loading oocytes, respectively. ㆍThe efficiency of freezing method and loading convenience were orderly higher in OPS, NLS and EMG. The optimal capacity per vessel was orderly lowered in NLS, EMG and OPS, respectively. ㆍAfter oocytes were warmed, the recovery rate, morphology and rate of development were orderly higher in OPS, NLS and EMG, respectively. ㆍIn conclusion, OPS has the advantages of achieving a little more survival and preserving results than other two loading methods.