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

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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.