用于小鼠基因操作的复杂靶向载体在酵母中的快速构建。
Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse.
作者信息
Storck T, Krüth U, Kolhekar R, Sprengel R, Seeburg P H
机构信息
Center for Molecular Biology (ZMBH), University of Heidelberg, Germany.
出版信息
Nucleic Acids Res. 1996 Nov 15;24(22):4594-6. doi: 10.1093/nar/24.22.4594.
Abstract
Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.
摘要
用于胚胎干细胞（ES细胞）的靶向载体通常包含一段大于7 kb的小鼠基因片段，其中插入了新霉素基因用于靶向事件的阳性选择。更复杂的靶向载体携带额外的遗传元件（如lacZ、loxP、点突变）。在这里，我们利用酵母中的同源重组构建靶向载体，用于将遗传元件（GEs）整合到小鼠基因中。GEs精确插入克隆在大肠杆菌/酵母穿梭载体中的小鼠基因片段的任何位置，由位于GEs两侧的短重组臂（RAs）引导。通过这种方式，可以相当轻松且快速地构建复杂的靶向载体，无需为寻找合适的限制性酶切位点进行广泛的基因图谱绘制。
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