Control of endogenous gene expression in the mouse

Several techniques have been developed to probe gene function through either loss or gain of function genetics in mice, such as conventional/conditional knock-outs and transgenics. However, cell lethality has limited the successful implementation of knockout technologies for some genes. In vivo knockdown approaches relying on tet-regulated siRNA expression may be able to address this problem, but siRNA development has proved challenging for some genes, and the degree of repression by RNA interference varies. Here, we present an alternative approach to achieving control of endogenous gene expression, and apply this system to the major DNA methyltransferase, Dnmt1.;Our approach to controlling endogenous gene expression employs a novel transcriptional regulatory system involving three binary systems, lacI, tetR and gal4, which allow for the reversible up- and down-regulation of a target gene from its endogenous locus. We engineered a number of modifications into both the lac operator and repressor, which significantly improved the repression as compared to wild type (WT) lac and tet systems. We introduced lac and tet operator sequences together with gal4 DBSs (DNA binding sequence) into the endogenous Dnmt1 promoter through gene-targeting, without noticeably altering promoter activity in the absence of the trans-elements. Endogenous Dnmt1 expression was successfully up and down regulated in a reversible temporo-spatial manner in mice. This technology was potent enough to reproduce the embryonic lethal phenotype of genetic knock-out and to attenuate transcription elongation, and the lethal phenotype was rescued by IPTG treatment. This establishes the first paradigm of experimental rescue of an embryonic lethal phenotype in loss-of-function genetics, and holds great promise for genes for which in vivo characterization have been limited due to a lethal phenotype.;The present study explores the possibility of controlling eukaryotic gene expression through the institution of a physical access to the endogenous promoter, and establishing a proof of principle for the methodology with Dnmt1, a gene for which the use of conventional/conditional knock-outs and transgenic approaches have been limited due to embryonic or cell lethality. In principle, this approach should be applicable to other genes and other organisms.