1. Epitope-tagging Math5 and Pou4f2: new tools to study retinal ganglion cell development in the mouse.
- Author
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Fu X, Kiyama T, Li R, Russell M, Klein WH, and Mu X
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Blotting, Southern, Fluorescent Antibody Technique, Gene Expression Regulation, Developmental genetics, Gene Expression Regulation, Developmental physiology, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Mice, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Transcription Factor Brn-3B genetics, Transcription Factor Brn-3B metabolism, Basic Helix-Loop-Helix Transcription Factors physiology, Epitopes genetics, Homeodomain Proteins physiology, Nerve Tissue Proteins physiology, Retinal Ganglion Cells metabolism, Transcription Factor Brn-3B physiology
- Abstract
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. Here, we overcome these limitations by constructing genetically engineered alleles for Math5 and Pou4f2, two genes required for retinal ganglion cell (RGC) development. Sequences encoding a peptide epitope from haemagglutinin (HA) were added to Math5 or Pou4f2 in frame to generate Math5(HA) and Pou4f2(HA) alleles. We demonstrate that the tagged alleles recapitulated the wild-type expression patterns of the two genes, and that the tags did not interfere with the function of the cognate proteins. In addition, by co-staining, we found that Math5 and Pou4f2 were transiently co-expressed in newly born RGCs, unequivocally demonstrating that Pou4f2 is immediately downstream of Math5 in RGC formation. The epitope-tagged alleles provide new and useful tools for analyzing gene regulatory networks underlying RGC development.
- Published
- 2009
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