title: A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0 Xenopus embryos and tadpoles.
abstract: The ability to express exogenous reporter genes in intact, externally developing embryos, such as Xenopus, is a powerful tool for characterizing the activity of cis-regulatory gene elements during development. Although methods exist for generating transgenic Xenopus lines, more simplified methods for use with F0 animals would significantly speed the characterization of these elements. We discovered that injecting 2-cell stage embryos with a plasmid bearing a ϕC31 integrase-targeted attB element and two dual β-globin HS4 insulators flanking a reporter transgene in opposite orientations relative to each other yielded persistent expression with sufficiently high penetrance for characterizing the activity of the promoter without having to coinject integrase RNA. Expression began appropriately during development and persisted into swimming tadpole stages without perturbing the expression of the cognate endogenous gene. Coinjected plasmids having the same elements but expressing different reporter proteins were reliably coexpressed within the same cells, providing a useful control for variations in injections between animals. To overcome the high propensity of these plasmids to undergo recombination, we developed a method for generating them using conventional cloning methods and DH5α cells for propagation. We conclude that this method offers a convenient and reliable way to evaluate the activity of cis-regulatory gene elements in the intact F0 embryo.
authors.affiliation: Department of Biological Sciences and the Center for Neuroscience Research, University at Albany, State University of New York, 1400 Washington Avenue, Albany, NY 12222, USA. Electronic address: cwang3@albany.edu.
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Biological Sciences and the Center for Neuroscience Research, University at Albany, State University of New York, 1400 Washington Avenue, Albany, NY 12222, USA. Electronic address: bgs86@albany.edu.
authors.forename: Ben G
authors.initials: BG
authors.lastname: Szaro
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D019426:Integrases / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014982:Xenopus laevis / Q000196:embryology* / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; D019426:Integrases
keywords: Cis-regulatory gene elements; HS4 insulator; Xenopus laevis; attB element