Epitope-tagging Math5 and Pou4f2: new tools to study retinal ganglion cell development in the mouse.
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.
Fu
Xueyao
X
Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA.
Kiyama
Takae
T
Li
Renzhong
R
Russell
Mark
M
Klein
William H
WH
Mu
Xiuqian
X
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015139:Blotting, Southern; D000939:Epitopes / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D050820:Transcription Factor Brn-3B / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D051792:Basic Helix-Loop-Helix Proteins; D000939:Epitopes; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D050820:Transcription Factor Brn-3B; C117638:Atoh7 protein, mouse; C085559:Pou4f2 protein, mouse