Li Z W, Stark G, Götz J, Rülicke T, Gschwind M, Huber G, Müller U, Weissmann C
Institut für Molekularbiologie der Universität Zürich, Switzerland.
Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6158-62. doi: 10.1073/pnas.93.12.6158.
Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.
通过在小鼠胚胎干细胞（ES细胞）中与合适的靶向载体进行同源重组，已产生了长达20kb的基因破坏和缺失。由于我们无法通过经典技术在小鼠淀粉样前体蛋白基因中获得约200kb的缺失，因此我们采用了一些策略，包括在通过同源重组要删除的区域的上游和下游插入loxP位点，并通过将Cre表达载体导入ES细胞来诱导loxP侧翼区域的切除。在第一种方法中，loxP序列分两步插入，每一步之后，分离并鉴定ES细胞克隆。loxP侧翼序列的缺失在第三步通过引入cre基因来完成。在第二种方法中，将含有上游loxP盒的ES细胞与下游loxP靶向载体和Cre表达质粒同时进行电穿孔。获得了能产生可将缺失的淀粉样前体蛋白等位基因进行种系传递的嵌合小鼠的ES细胞。
