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Successful inhibition of Dnmt1 expression in bovine using RNA interference.
Author(s) -
Sosa Hector Eduardo,
LaMarre Jonathan,
King Allan
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb198
Subject(s) - rna interference , reprogramming , dnmt1 , biology , gene knockdown , epigenetics , dna methylation , methylation , microbiology and biotechnology , gene expression , gene silencing , small hairpin rna , gene , rna , genetics
Epigenetic modifications, such as methylation are essential for normal mammalian development. Dnmt1 is the predominant and most active methylase, which is responsible for copying methylation patterns after replication. During early development, Dnmt1 is highly regulated to support passive demethylation and normal reprogramming. In somatic cells, Dnmt1 is localized in the nucleus and its introduction into cloned embryos is believed to be the main limiting factor for proper reprogramming of the donor genome. The aim of this study was to transiently suppress Dnmt1 by RNA interference (RNAi) to investigate the role of Dnmt1 during early development and its effect on nuclear transfer technology. RNA and protein extracts were collected from fibroblasts treated with control or Dnmt1 RNAi. Real time RT‐PCR revealed a 93.4% reduction of Dnmt1 transcript levels. Consistently, Western blot analysis showed a protein decrease of up to 98.8%. Gene expression of housekeeping genes, GAPD, ACTB and lamin A/C, were not affected after RNAi treatment however, expression of HPRT1, H19, MMP1 and SCYA genes was increased. This is the first report to demonstrate a specific and highly effective knockdown of bovine Dnmt1 by RNAi at both mRNA and protein levels. Dnmt1 RNAi could be a useful tool to better understand epigenetic reprogramming events as well as syndromes related to abnormal methylation patterns. (Funding by CONACyT & NSERC)