title: Endogenous tagging of the murine transcription factor Sox5 with hemaglutinin for functional studies.
abstract: Gene expression is usually studied at the transcript level rather than at the protein level due to the lack of a specific and sensitive antibody. A way to overcome this is to fuse to the protein of interest an immunoreactive tag that has well-characterized antibodies. This epitope tagging approach is often used for in vitro experiments but for in vivo studies, the success rate of protein tagging has not been extensively analyzed and our study seeks to cover the void. A small epitope, hemaglutinin derived from the influenza virus was used to tag a transcription factor, Sox5 at the N-terminal via homologous recombination in the mouse. Sox5 is part of the Sry-related high-mobility-group box gene family and plays multiple roles in essential biological processes. Understanding of its molecular function in relation to its biological roles remains incomplete. In our study, we show that the longer isoform of Sox5 can be tagged endogenously with hemaglutinin without affecting its biological function in vivo. The tagged protein is easily and specifically detected with an anti-hemaglutinin antibody using immunohistochemistry with its expression matching the endogenous expression of Sox5. Immunoprecipitation of Sox5 was also carried out successfully using an anti-hemaglutinin antibody. The transgenic line generated from this study is predicted to be useful for future experiments such as co-immunoprecipitation and chromatin immunoprecipitation, allowing the further understanding of Sox5. Lastly, this approach can be easily employed for the investigation of other transcription factors and proteins in vivo to overcome technical limitations such as antibody cross-reactivity and to perform isoform-specific studies.
authors: Stem Cell and Developmental Biology, Genome Institute of Singapore, 60 Biopolis Street, Singapore, 138672, Singapore.
authors: Wenqing Jean
authors: WJ
authors: Lee
authors: Petra
authors: P
authors: Kraus
authors: Thomas
authors: T
authors: Lufkin
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D060005:Genotyping Techniques; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D047468:Immunoprecipitation / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008845:Microinjections; D009975:Orthomyxoviridae / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055755:SOXD Transcription Factors / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D020033:Protein Isoforms; D055755:SOXD Transcription Factors; C089186:Sox5 protein, mouse