Development of a positive-negative selection procedure for gene targeting in fish cells
Development of a positive-negative selection procedure for gene targeting in fish cells
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DOI：
10.1016/s0044-8486(01)00811-0
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发表时间：
2002-11-15
期刊：
影响因子：
4.5
通讯作者：
Schartl, M
中科院分区：
文献类型：
--
作者：
Chen, SL;Hong, YH;Schartl, M
基因打靶技术是分析克隆基因功能和产生定点整合外源基因的转基因动物的有力工具。为了在FISH中发展这一技术，构建了正负选择(PNS)和同源重组载体，并检测了它们在FISH细胞中的表达。PNS的载体(PNK)由新霉素抗性基因(Neo)作为阳性选择标记基因和单纯疱疹病毒胸苷激酶(Tk)基因作为阴性选择标记基因组成。用G418对转染线性化PNK载体的鲤鱼丘疹上皮瘤细胞(EPC)进行阳性筛选可获得350个克隆，而G418和更昔洛韦(GC)双重筛选可使细胞几乎完全死亡，证明PNS方法在鱼类细胞中是有效的。同源重组载体除含有neo和tk基因外，还含有剑尾鱼黑色素瘤受体激酶(X mrk(Y))基因作为同源序列。建立了同源重组载体转染和药物筛选的条件。在验证了同源重组载体在EPC细胞中表达的可行性后，首次在剑尾蛇黑色素瘤细胞系PSM中进行了基因打靶实验。靶向载体-转染体的正负选择导致该特定细胞系的低丰度。对PSM细胞低浓集的原因进行了讨论。(C)2002 Elsevier Science B.V.保留所有权利。
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.