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

Tucker, Kerry L.; Talbot, Dale; Lee, Min Ae; Leonhardt, Heinrich; Jaenisch, Rudolf

doi:10.1073/pnas.93.23.12920

Cited by 82 publications

(50 citation statements)

References 37 publications

(39 reference statements)

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“…Our data unequivocally show that the 5Ј-end of the Dnmt1 gene contains three additional in-frame ATGs in all mouse DNAs tested (see ATG1-3 in Fig. 1) and thus confirm our previous observation (15). As shown in Fig.…”

Section: Resultssupporting

confidence: 78%

“…The Mouse Dnmt1 Gene Codes for at Least Two MTase Isoforms-Recent reports of additional 5Ј-cDNA sequences of the mouse Dnmt1 gene differed by one frameshift, which alters the number of possible upstream ATGs (15,25). Because this discrepancy has potential consequences for the structure of the N-terminal, regulatory domain, we sequenced the corresponding genomic DNA of the mouse Dnmt1 gene.…”

Section: Resultsmentioning

confidence: 99%

“…For these experiments, we chose Dnmt1 null ES cells, which do not express MTase and have a hypomethylated genome but can be rescued by transfection with a Dnmt1 minigene (see pMT50, Fig. 1A) (15). We systematically mutagenized each of the four possible in-frame ATGs (ATG 3 TTG) of the Dnmt1 minigene construct and tested them in null ES cells.…”

Section: Resultsmentioning

confidence: 99%

“…Vectors and DNA Sequencing-Site-directed mutations were introduced in the Dnmt1 minigene using the previously described plasmid pMT50 (15). The ATG mutant constructs pMT⌬1,2 (mutated ATG1,2), pMT⌬3 (mutated ATG3), and pMT⌬1,2,3 (mutated ATG1,2,3) were generated using standard PCR-mediated site-directed mutagenesis with mutant primers overlapping ATG1,2 or ATG3: ⌬1,2-upper primer, 5Ј-GCCTCCGTTGCGCGCtTGCGCACTCCCTTCGGGCATAGCtTGG-TCTTCCCCCACTCT-3Ј; ⌬3-upper primer, 5Ј-CCTGCAAGtTGCCAGC-GCGAACAGC-3Ј; ⌬1,2-lower primer, 5Ј-AGAGTGGGGGAAGACCAaGCTATGCCCGAAGGGAGTGCGCAaGCGCGCAACGGAGGC-3Ј; and ⌬3-lower primer, 5Ј-GCTGTTCGCGCTGGCAaCTTGCAGG-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…Tissues-Dnmt1 null ES cells die upon induction of differentiation and cannot form teratomas (15). To test whether MTase ATG4 can rescue the differentiation capacity of these cells, we injected MTase ATG4 -transfected ES cells in the flank of 129/sv male mice to generate teratomas.…”

Section: The Shorter Mtase Isoform Can Substitute For the Longer Formmentioning

confidence: 99%

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A Short DNA Methyltransferase Isoform Restores Methylation In Vivo

Gaudet¹,

Talbot²,

Leonhardt³

et al. 1998

Journal of Biological Chemistry

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Two murine DNA methyltransferase isoforms (MTases) have been observed, a longer form in somatic and embryonic stem (ES) cells and a shorter form in oocytes and preimplantation embryos. While the longer MTase is associated with maintenance methyltransferase activity in replicating cells, little is known about the shorter form. We present genetic and biochemical evidence that both isoforms are expressed from the same Dnmt1 gene by using different translation initiation sites in exons 1 and 4. We further demonstrate that the shorter isoform can functionally rescue Dnmt1 null ES cells that have a hypomethylated genome. These rescued ES cells differentiate in vivo into a variety of cell types, unlike the Dnmt1 null ES cells that die upon induction of differentiation. These results show that the shorter isoform can substitute for the longer maintenance MTase in ES and differentiated cells. Our data further indicate that the shorter MTase isoform found in oocytes is fully functional in vivo and may play an active role in the regulation of DNA methylation and the establishment of imprinting patterns.

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Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: The Shorter Mtase Isoform Can Substitute For the Longer Formmentioning

confidence: 99%

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A Short DNA Methyltransferase Isoform Restores Methylation In Vivo

Gaudet¹,

Talbot²,

Leonhardt³

et al. 1998

Journal of Biological Chemistry

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Gene knockout and transgenic technologies in risk assessment: The next generation

Rosenberg

1997

Mol. Carcinog.

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Transgenic and knockout mice have been proposed as substitutes for one of the standard 2-yr rodent assays. The advantages of using genetically engineered mouse models is that fewer mice are needed, the time to develop disease is greatly reduced, and the mice are predisposed to developing cancer by virtue of gain or loss of functions. The models currently being used have yielded a large amount of data and have proved to be informative for risk assessment; however, they are still far from ideal. In fact, they inherently do not reflect the complexity of mutation and carcinogenesis in humans. Recent advances in technology and the creation of new knockout mice may produce more useful and more sensitive models. This review covers two recent advances in technology--inducible and regulatable gene expression and targeted genetic modifications in the genome--that will allow us to make better models. I also discuss new gene deletion and transgenic mouse models and their potential impact on risk-assessment assays. These models are presented in the context of four basic components or events that occur in the multistep process leading to cancer: maintenance of gene expression patterns, genome stability and DNA repair, cell-cell communication and signaling, and cell-cycle regulation. Finally, surrogate markers and utility in risk assessment are also discussed. This review is meant to stimulate further discussion in the field and to generate excitement about working toward the next generation of risk-assessment models.

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Gene-trap-based target site for Cre-mediated transgenic insertion

Hardouin¹,

Nagy

2000

genesis

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Summary: There is an increasing need for tissue‐specific gene expression regulatory elements to study normal and disease development in the mouse. However, the cloning and characterization of these elements are time‐consuming and costly. Thus, there is a particular need to be able to identify gene expression patterns without having to clone the promoter elements. Gene‐trap strategies identify expression patterns assigned for endogenous genes using reporters, such as LacZ (Gossler et al., 1989; Skarnes, 1990) or green fluorescent protein (GFP) (Ishida and Leder, 1999; Zheng and Hughes, 1999). The gene‐trap vector randomly inserts into the genome and “steals” regulatory elements for the reporter. Here we describe an improved gene‐trap strategy, which allows an efficient Cre recombinase‐mediated insertion of any transgene into the trapped loci as a post‐integrational modification and links the expression of the transgene to that of the reporter. genesis 26:245–252, 2000. © 2000 Wiley‐Liss, Inc.

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

Cited by 82 publications

References 37 publications

A Short DNA Methyltransferase Isoform Restores Methylation In Vivo

A Short DNA Methyltransferase Isoform Restores Methylation In Vivo

Gene knockout and transgenic technologies in risk assessment: The next generation

Gene-trap-based target site for Cre-mediated transgenic insertion

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