Tracing Gene Expression Through Detection of β-galactosidase Activity in Whole Mouse Embryos
| Autor: | Ana I R Learte, Cristina Sánchez-Camacho, Emma Muñoz-Sáez, Miguel A Marchena, María Antonia Cid, Iván Rodríguez-Martín, Maria Blanco |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
General Immunology and Microbiology Chemistry General Chemical Engineering General Neuroscience lac operon Embryo Galactosidase activity medicine.disease_cause General Biochemistry Genetics and Molecular Biology Staining Cell biology 03 medical and health sciences 030104 developmental biology Gene expression medicine Gene Developmental biology Escherichia coli |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/57785 |
| Popis: | The Escherichia coli LacZ gene, encoding β-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, β-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of β-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for β-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning. |
| Databáze: | OpenAIRE |
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