鱼类的胚胎干细胞(英文)

胚胎干细胞 (ES)是未分化的细胞培养物 ,来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下 ,ES细胞能分化成各种细胞类型 ,包括生殖细胞。这样 ,ES细胞就提供了一个有效的纽带 ,将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前 ,ES细胞技术仅见之老鼠 ,因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类 ,人们已做了大量的尝试。我们以青 (Oryziaslatipes)作为建立鱼类ES细胞技术的模式 ,通过建立并应用无滋

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

Efficiency of constructed wetlands in decontamination of water polluted by heavy metals

A twin-shaped constructed wetland (CW) comprising a vertical flow (inflow) chamber with Cyperus alternifolius followed by a reverse-vertical flow (outflow) chamber with Villarsia exaltata was assessed for decontamination of artificial wastewater polluted by heavy metals. After application of Cd, Cu, Pb, Zn over 150 days, together with Al and Mn during the final 114 days, no heavy metals with the exception of Mn could be detected in either the drainage zone at the bottom, shared by both chambers, or in the effluent. The inflow chamber was, therefore, seen to be predominantly responsible for the decontamination process of more toxic metal species with final concentrations far below WHO drinking-water standards. About one-third of the applied Cu and Mn was absorbed, predominantly by lateral roots of C. alternifolius. Lower accumulation levels were observed for Zn (5%), Cd (6%), Al (13%). and Pb (14%). Contents of Cd, Cu, Mn, and Zn in soil were highest in top layer, while Al and Pb were evenly distributed through the whole soil column. Metal species accumulating mainly in the top layer can be removed mechanically. A vertical flow CW with C. alternifolius is an effective tool in phytoremediation for treatment of water polluted with heavy metals. (C) 2002 Elsevier Science B.V. All rights reserved.