Development of a positive-negative selection procedure for gene targeting in fish cells
文献类型：期刊论文
| 作者 | Chen, SL; Hong, YH; Schartl, M |
| 刊名 | AQUACULTURE
 |
| 出版日期 | 2002-11-15 |
| 卷号 | 214期号:1-4页码:67-79 |
| 关键词 | fish gene targeting homologous recombination vector positive-negative selection Xiphophorus melanoma receptor kinase gene (X mrk) |
| ISSN号 | 0044-8486 |
| 通讯作者 | Hong, YH, Natl Univ Singapore, Dept Biol Sci, 10 Kent Ridge Crescent, Singapore 119260, Singapore |
| 中文摘要 | 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. |
| 英文摘要 | 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. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 学科主题 | Fisheries; Marine & Freshwater Biology |
| 类目[WOS] | Fisheries ; Marine & Freshwater Biology |
| 研究领域[WOS] | Fisheries ; Marine & Freshwater Biology |
| 关键词[WOS] | MEDAKA ORYZIAS-LATIPES ; EMBRYONIC STEM-CELLS ; GERM-LINE CHIMERAS ; HOMOLOGOUS RECOMBINATION ; MELANOMA ; XIPHOPHORUS ; ESTABLISHMENT ; ZEBRAFISH ; CULTURES ; EXPRESSION |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000178793800005 |
| 公开日期 | 2010-10-13 |
| 源URL | [http://ir.ihb.ac.cn/handle/152342/9786]  |
| 专题 | 水生生物研究所_中科院水生所知识产出（2009年前）_期刊论文 |
| 作者单位 | 1.Univ Wurzburg, Bioctr, Physiol Chem 1, D-97074 Wurzburg, Germany 2.Chinese Acad Fisheries Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China 3.Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China |
| 推荐引用方式 GB/T 7714 | Chen, SL,Hong, YH,Schartl, M. Development of a positive-negative selection procedure for gene targeting in fish cells[J]. AQUACULTURE,2002,214(1-4):67-79. |
| APA | Chen, SL,Hong, YH,&Schartl, M.(2002).Development of a positive-negative selection procedure for gene targeting in fish cells.AQUACULTURE,214(1-4),67-79. |
| MLA | Chen, SL,et al."Development of a positive-negative selection procedure for gene targeting in fish cells".AQUACULTURE 214.1-4(2002):67-79. |
入库方式： OAI收割
来源：水生生物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。