Establishment of genetically modified mouse libraries using Cre/loxP gene trap

本研究的目的是评估利用Cre/lop诱捕系统建立转基因小鼠文库的可能性。该研究还旨在生产以各种方式表达Cre重组酶的转基因小鼠系，从而实现第二代基因敲除。1) ES细胞的基因诱捕及诱捕基因的鉴定我们制备了在PGK启动子下含有polyA - less Puromycin耐药基因的诱捕载体。同时，设计该载体诱捕某内源基因的启动子，表达Cre重组酶。利用这个载体，我们获得了600个被困的克隆体。利用3′-RACE对捕获的克隆进行分析，鉴定出9条已知的基因2 EST序列。其他序列来自未知的基因。该载体被证明可以有效地捕获胚胎干细胞中沉默的基因。2)利用捕获的胚胎干细胞制备嵌合小鼠。嵌合小鼠是从捕获的胚胎干细胞系中产生的，目前已经建立了10多个转基因小鼠系。这些基因被破坏的小鼠的表型目前正在研究中。用GFP转基因小鼠检测这些捕获小鼠的Cre重组酶的表达，GFP转基因小鼠在表达Cre的器官中变为绿色。为了确定Cre表达器官（或细胞）的定位，我们制作了报告“绿色小鼠”，当Cre重组酶重组转基因时，它们会变成荧光绿色。

The purpose of this study is to estimate the possibility of establishing genetically modified mouse libraries using Cre/lop trap system. The research also aimed to produce transgenic mouse lines that express Cre recombinase in various ways to enable a second generation of gene knockout.1) Gene trap in ES cells and identification of the trapped geneWe have produced trap vector that contains polyA less Puromycin resistant gene under the PGK promoter. At the same time, the vector was designed to trap a promoter of a certain endogenous gene and express Cre recombinase. Using this vector, we have obtained 600 trapped clones. After the analysis of the trapped clones using 3'-RACE, we identified 9 known gene 2 EST sequences. Other sequences were originated from unknown genes. The vector was shown to be effective to trap genes which are silent in ES cells.2) Prodction of Chimeric mouse from trapped ES cells.Chimeric mice are produced from the trapped ES cell lines and at the present more than 10 genetically modified mouse lines are established. The phenotypes of these gene-disrupted mice are now under investigations. The expression of the Cre recombinase from these trapped mice are also examined using GFP transgenic mice which becomes green in organs where Cre is expressed.In order to identify the localization of Cre expressing organs (or cells), we produced reporter "green mice" which turns fluorescent green when Cre recombinase recombine the transgene.