Insights into the role of DNA methylation in murine embryonic stem cells using a modified tetracycline-inducible gene expression system.
Murine embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mouse blastocyst. Their ability to differentiate into diverse cell types in vitro has served well as a model of early embryonic developmental processes. A powerful strategy for studying the role of key genes during this time is to be able to regulate their expression in a dose-dependent manner. Here, we outline our experience of applying the "tetracycline-inducible system" to study gene function during ES cell differentiation and describe a number of key modifications designed to overcome the central problem of transgene silencing. Finally, we demonstrate how this approach can yield insights into the role of DNA methylation during ES cell differentiation by studying the function of two genes: DNMT3A, involved in the de novo methylation of cytosine bases, and MBD2b, a methyl-dependent co-repressor and putative cytosine demethylase.
John Hughes Bennett Laboratories, Western General Hospital, Edinburgh, Scotland, EH4 2XU, UK.
Timothy
T
Chevassut
Bing
B
Lim
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D002999:Clone Cells; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D051379:Mice; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology; D000097584:DNA Methyltransferase 3B
D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D004268:DNA-Binding Proteins; D013752:Tetracycline; D000097584:DNA Methyltransferase 3B; C000715535:Dnmt3a protein, mouse; C115368:MBD2 protein