Development of systems for cell culture and expression vector to generate transgenic marine invertegra

1.调控海胆基因表达的顺式活性元件的鉴定：通过报告实验，在海胆Ars基因转录起始点附近检测到几个调控海胆Ars基因转录的序列元件。其中SOX结合位点间-168bp到-162bp之间最为显著。检测到-186bp左右抑制启动子活性的顺式元件。绝缘体元件在基因转移实验中的有效性：海胆Ars基因上游存在的绝缘体元件在果蝇、小鼠和人类的细胞中也显示了绝缘体活性。珍珠牡蛎外套膜上皮细胞培养体系：建立了珍珠牡蛎外套膜上皮细胞的原代培养体系，尽管珍珠牡蛎外套膜上皮细胞在体外不能增殖。海胆胚胎细胞的培养：虽然我们成功地建立了海胆幼体细胞的体外培养，但由于难以防止污染真菌的增殖，未能培养超过3天。

1. Identification of cis-active elements that regulate expression of sea urchin gene :Several sequence elements that regulate transcription of the sea urchin Ars gene were detected by reporter assay around its transcription start site. Among them between Sox-binding sitebetween -168bp and -162bp is most prominent element. Around -186bp cis-element that represses the promoter activity was detected.2. Validity of insulator element in gene transfer experiments :An insulator element present at the upstream of the sea urchin Ars gene showed insulator activity also in cells of Drosophila, mouse and humane3. Cell culture system for mantle epitermal cells of pearl oyster :Primary cell culture system for mantle epitermal cells of pearl oyster was established, though they do not proliferate in vitro.4. Culturing of sea urchin embryonic cells :Though we succeeded in establishing in vitro culture of cells from sea urchin pluteus larvae, we failed to culture them beyond three days because of difficulty in preventing proliferation of contaminating fungi.