Enhanced gene targeting frequency in ES cells with low genomic methylation levels

Increased methylation in promoter/enhancer regions typically results in transcriptional downregulation. The direct correlation between gene expression and homologous recombination (HR) is also widely acknowledged, and suggests that actively transcribed, hypomethylated targets may be more accessible to the HR machinery. Consistent with this hypothesis, we report that DNA methyltransferase 1 (Dnmt1)-knockout ES cells show a 2-fold increase in gene targeting frequency. However, the use of hypomethylated targeting vectors or the ectopic expression of a putative DNA demethylase did not enhance targeting frequency. These observations are discussed in the context of devising more efficient targeting protocols by transiently modifying genomic methylation levels.