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Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

Item Type:Article
Title:Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene
Creators Name:Tucker, K.L. and Talbot, D. and Lee, M.A. and Leonhardt, H. and Jaenisch, R.
Abstract:Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS/PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.
Keywords:Amino Acid Sequence, Antibodies, Base Sequence, Cell Line, DNA (Cytosine-5-)-Methyltransferase, DNA Methylation, DNA Primers, Exons, Genetic Complementation Test, Genetic Vectors, Molecular Sequence Data, Moloney Murine Leukemia Virus, Nucleic Acid Sequence Homology, Open Reading Frames, Peptide Fragments, Recombinant Proteins, Stem Cells, Transfection, Animals, Mice
Source:Proceedings of the National Academy of Sciences of the United States of America
ISSN:0027-8424
Publisher:National Academy of Sciences (U.S.A.)
Volume:93
Number:23
Page Range:12920-12925
Date:12 November 1996
Official Publication:http://www.pnas.org/content/93/23/12920.abstract
PubMed:View item in PubMed

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