title: A transient transgenic RNAi strategy for rapid characterization of gene function during embryonic development.
abstract: RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for "transient transgenic" knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method.
authors.affiliation: Genetics Division, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Bjork
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Shannon W
authors.initials: SW
authors.lastname: Davis
authors.forename: Haiyan
authors.initials: H
authors.lastname: Qiu
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Saunders
authors.forename: Peter
authors.initials: P
authors.lastname: Sandy
authors.forename: Stuart
authors.initials: S
authors.lastname: Orkin
authors.forename: Sally A
authors.initials: SA
authors.lastname: Camper
authors.forename: David R
authors.initials: DR
authors.lastname: Beier
mesh_terms: D000818:Animals; D001483:Base Sequence; D002972:Cleft Palate / Q000235:genetics*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics; D015509:Developmental Biology / Q000379:methods; D004195:Disease Models, Animal; D053595:Embryonic Stem Cells / Q000166:cytology; D018507:Gene Expression Regulation, Developmental; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D034622:RNA Interference; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C521133:Prdm16 protein, mouse; D014157:Transcription Factors