title: Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus.
abstract: We have developed a method of injecting bacterial artificial chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.
authors.affiliation: Department of Biology, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Margaret B
authors.initials: MB
authors.lastname: Fish
authors.forename: Takuya
authors.initials: T
authors.lastname: Nakayama
authors.forename: Robert M
authors.initials: RM
authors.lastname: Grainger
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D019076:Transgenes; D014981:Xenopus / Q000196:embryology / Q000235:genetics*; D029867:Xenopus Proteins / Q000235:genetics
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D029867:Xenopus Proteins; C109416:ATOH7 protein, Xenopus; C524416:Rax protein, Xenopus
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