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Development of a positive-negative selection procedure for gene targeting in fish cells
Songlin Chen; Yunhan Hong; Manfred Schartl; Hong, YH, Natl Univ Singapore, Dept Biol Sci, 10 Kent Ridge Crescent, Singapore 119260, Singapore
2002
Source PublicationAQUACULTURE
Volume214Issue:1-4Pages:67-79
AbstractThe 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.
KeywordFish Gene Targeting Homologous Recombination Vector Positive-negative Selection Xiphophorus Melanoma Receptor Kinase Gene (x Mrk)
DepartmentUniv Wurzburg, Bioctr, Physiol Chem 1, D-97074 Wurzburg, Germany; Chinese Acad Fisheries Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China
Subject AreaFisheries ; Marine & Freshwater Biology
Indexed BySCI
WOS IDWOS:000178793800005
Citation statistics
Cited Times:18[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/152342/9786
Collection期刊论文
Corresponding AuthorHong, YH, Natl Univ Singapore, Dept Biol Sci, 10 Kent Ridge Crescent, Singapore 119260, Singapore
Recommended Citation
GB/T 7714
Songlin Chen,Yunhan Hong,Manfred Schartl,et al. Development of a positive-negative selection procedure for gene targeting in fish cells[J]. AQUACULTURE,2002,214(1-4):67-79.
APA Songlin Chen,Yunhan Hong,Manfred Schartl,&Hong, YH, Natl Univ Singapore, Dept Biol Sci, 10 Kent Ridge Crescent, Singapore 119260, Singapore.(2002).Development of a positive-negative selection procedure for gene targeting in fish cells.AQUACULTURE,214(1-4),67-79.
MLA Songlin Chen,et al."Development of a positive-negative selection procedure for gene targeting in fish cells".AQUACULTURE 214.1-4(2002):67-79.
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