UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Publication Date

2-2-2006

Document Type

Article

Subjects

3' Untranslated Regions; Animals; Base Sequence; DNA Primers; Female; Fertilization; Gene Expression Regulation, Enzymologic; Mice; Mice, Knockout; Mice, Transgenic; Nucleic Acid Conformation; Ovum; Phenotype; Polymerase Chain Reaction; RNA; RNA Interference; RNA Polymerase II; Superoxide Dismutase

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)-expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)-the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.

Rights and Permissions

Citation: PLoS Genet. 2006 Jan;2(1):e10. Epub 2006 Jan 27. Link to article on publisher's site

DOI of Published Version

10.1371/journal.pgen.0020010

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

PLoS genetics

PubMed ID

16450009

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