title: Spatiotemporal control of embryonic gene expression using caged morpholinos.
abstract: Embryonic development depends on spatial and temporal control of gene function, and deciphering the molecular mechanisms that underlie pattern formation requires methods for perturbing gene expression with similar precision. Emerging chemical technologies can enable such perturbations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to photo-inactivate genes in zebrafish embryos with spatiotemporal control. This chapter describes general principles for cMO design and methods for cMO assembly in three steps from commercially available reagents. Experimental techniques for the microinjection and photoactivation of these reagents are described in detail, as well as the preparation and application of caged fluorescein dextran (cFD) for labeling irradiated cells. Using these protocols, cMOs can be effective tools for functional genomic studies in zebrafish and other model organisms.
authors.affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford,California, USA.
authors.forename: Ilya A
authors.initials: IA
authors.lastname: Shestopalov
authors.forename: James K
authors.initials: JK
authors.lastname: Chen
mesh_terms: D000818:Animals; D019521:Body Patterning / Q000235:genetics*; D003432:Cross-Linking Reagents / Q000528:radiation effects; D019793:Fluorescein / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D007814:Larva / Q000235:genetics; D008845:Microinjections / Q000379:methods; D060172:Morpholinos / Q000138:chemical synthesis / Q000302:isolation & purification / Q000528:radiation effects*; D044366:Transition Temperature; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D060172:Morpholinos; D019793:Fluorescein