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X. Mu, X. Fu, T. Kiyama, C.-A. Mao, R. Li, M. Russell, W. H. Klein; Epitope-tagging Math5 And Pou4f2: New Tools to Study Retinal Ganglion Cell Development in the Mouse. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4005.
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© ARVO (1962-2015); The Authors (2016-present)
Although immunological detection of proteins is used extensively in retinal development, studies are often impeded because antibodies against crucial proteins cannot be generated or are not readily available. In order to overcome these limitations, we constructed three genetically engineered alleles for Math5 and Pou4f2 in which epitope tags were added.
The three alleles were generated by gene targeting in mouse ES cells and positive clones were identified by Southern blot hybridization. These three alleles are Math5HA, Math5HAEGFP and Pou4f2HA. Targeted Math5HA and Pou4f2HA ES cells were used for blastocyst injections and germline transmission to establish Math5HA and Pou4f2HA mouse lines. We further characterized the function of these alleles by histology and antibody staining.
Immunostaining of developing retinas with an anti-HA antibody demonstrated that the tagged alleles recapitulated the wild-type expression patterns of the two genes. Both Math5HA/HA and Pou4f2HA/HA mice were viable, fertile. Marker analysis indicated that retinal ganglion cells develop normally in both Math5HA/HA and Pou4f2HA/HA retinas. Development of other cell types were not affected either. We also show that the Math5HAEGFP allele can be used to monitor Math5 expression when embryonic stem cells were induced to differentiate into retinal neurons.
We generated three knock-in alleles in which the Math5 and Pou4f2 genes were epitope-tagged with HA or HA-EGFP sequences. The tags did not affect the normal function of the cognate proteins. These alleles should contribute substantially to an ever-expanding genetic toolbox from which a comprehensive understanding of retinal development will eventually emerge.
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