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PMID: 21179568 Published · epublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

A transient transgenic RNAi strategy for rapid characterization of gene function during embryonic development.

PloS one ·Vol. 5 ·No. 12 ·2010-12-16 ·Pages e14375

Bjork BC, Fujiwara Y, Davis SW, Qiu H, Saunders TL, Sandy P, Orkin S, Camper SA, Beier DR

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.

MeSH Terms
Animals Base Sequence Cleft Palate/genetics DNA Transposable Elements DNA-Binding Proteins/genetics Developmental Biology/methods Disease Models, Animal Embryonic Stem Cells/cytology Gene Expression Regulation, Developmental Lentivirus/genetics Mice Mice, Knockout Mice, Transgenic Molecular Sequence Data Mutation Oligonucleotide Array Sequence Analysis Phenotype RNA Interference Transcription Factors/genetics Transgenes
Chemicals
DNA Transposable Elements DNA-Binding Proteins Prdm16 protein, mouse Transcription Factors
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Bjork Bryan C
Genetics Division, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
Fujiwara Yuko
Davis Shannon W
Qiu Haiyan
Saunders Thomas L
Sandy Peter
Orkin Stuart
Camper Sally A
Beier David R
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Article Info
Journal
PloS one
Abbr.
PLoS One
ISSN
1932-6203
DOI
10.1371/journal.pone.0014375
Published
2010-12-16
Epub
2010-00-16
Pages
e14375
Language
English
Region
United States
NLM ID
101285081
PMCID
PMC3002952
Subset
IM
Grants
NIA NIH HHS · P30AG013283 · United States
NCI NIH HHS · P30 CA046592 · United States
NICHD NIH HHS · R01HD36404 · United States
NIDDK NIH HHS · DK34933 · United States
NIDCR NIH HHS · K12 DE014528 · United States
NCI NIH HHS · CA46592 · United States
NICHD NIH HHS · F32HD045066 · United States
NIMH NIH HHS · R01MH081187 · United States
NIA NIH HHS · P30 AG013283 · United States
NICHD NIH HHS · R37HD30428 · United States
NICHD NIH HHS · R01 HD036404 · United States
NIMH NIH HHS · R01 MH081187 · United States
NICHD NIH HHS · R01 HD034283 · United States
NICHD NIH HHS · R01HD34283 · United States
NIDCR NIH HHS · K12DE014528 · United States
NIAMS NIH HHS · AR20557 · United States
NICHD NIH HHS · R37 HD030428 · United States
NICHD NIH HHS · F32 HD045066 · United States
NIDDK NIH HHS · P30 DK034933 · United States
External Links
PubMed source DOI: 10.1371/journal.pone.0014375 PMC Full Text View journal details
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