Please use this identifier to cite or link to this item: https://doi.org/10.1016/S0044-8486(01)00811-0
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dc.titleDevelopment of a positive-negative selection procedure for gene targeting in fish cells
dc.contributor.authorChen, S.
dc.contributor.authorHong, Y.
dc.contributor.authorSchartl, M.
dc.date.accessioned2014-10-27T08:25:53Z
dc.date.available2014-10-27T08:25:53Z
dc.date.issued2002-11-15
dc.identifier.citationChen, S., Hong, Y., Schartl, M. (2002-11-15). Development of a positive-negative selection procedure for gene targeting in fish cells. Aquaculture 214 (1-4) : 67-79. ScholarBank@NUS Repository. https://doi.org/10.1016/S0044-8486(01)00811-0
dc.identifier.issn00448486
dc.identifier.urihttp://scholarbank.nus.edu.sg/handle/10635/100440
dc.description.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 mrkY) 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. © 2002 Elsevier Science B.V. All rights reserved.
dc.description.urihttp://libproxy1.nus.edu.sg/login?url=http://dx.doi.org/10.1016/S0044-8486(01)00811-0
dc.sourceScopus
dc.subjectFish
dc.subjectGene targeting
dc.subjectHomologous recombination vector
dc.subjectPositive-negative selection
dc.subjectXiphophorus melanoma receptor kinase gene (X mrk)
dc.typeArticle
dc.contributor.departmentBIOLOGICAL SCIENCES
dc.description.doi10.1016/S0044-8486(01)00811-0
dc.description.sourcetitleAquaculture
dc.description.volume214
dc.description.issue1-4
dc.description.page67-79
dc.description.codenAQCLA
dc.identifier.isiut000178793800005
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