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J.P. Marion, T. Fujimaki, H. Kitagawa, H. Sakuma, A. Murakami, Z.–Y. Huang, G. Inana; Construction of a Knock–Out Model of X–Arrestin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3730. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
X–arrestin is a retina–specific gene, which we have cloned by a subtractive cloning strategy. It is present specifically in the outer segments of red–, green–, and blue– cone photoreceptors in the human. X–arrestin is likely to have a function similar to that of rod arrestin. Arrestins are known to be signal quenchers of G–protein–coupled receptors, and rod photoreceptor arrestin desensitizes the light–activated phosphorylated rhodopsin. Thus, the function of X–arrestin is thought to be involved in the desensitization of light–activated cone opsins. The X–arrestin gene, consisting of 17 exons, has been isolated and characterized, and promoter studies of the gene have been conducted. The purpose of this study is to investigate the true function of X–arrestin in vivo by constructing and studying a knock–out mouse model of X–arrestin.
A DNA construct used for constructing the KO mouse model was prepared by replacing exons 12 and 13 of the X–arrestin gene with the neomycin resistance gene sequence and inserting the modified gene sequence into the pPNT vector. The construct was then transfected into embryonic stem (ES) cells. Successfully targeted ES cells were injected into mouse blastocysts to produce chimeras. Chimeras were crossed with normal mates to produce pups that may show germ line transmission and heterozygously targeted X–arrestin gene.
Transfection of ES cells with the KO construct yielded two lines of successfully targeted ES cells (#15 and #107). Injection of blastocysts with the #107 ES cells resulted in the production of 6 chimeras. One of the chimeras produced offspring with the Agouti phenotype, indicating successful germ line transmission.
The X–arrestin KO mouse model has been constructed, and germ line transmission has been confirmed. Confirmation of successful targeting of the X–arrestin gene in the pups is in progress.
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