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Catherine Clinton

Evaluation of DNMT-1 knockdown in adult bovine fibroblasts using RNA interference (RNAi)

Catherine Clinton, Todd Stroud, Charles Long and Mark Westhusin

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine,  Texas A&M University, College Station, Texas, USA

Catherine Clinton

Objective – To use RNA-interference (RNAi) to knock down DNA Methyltransferase-1 (DNMT-1) and artificially control DNMT-1 expression in bovine fibroblasts (BF). To evaluate and quantitate DNMT-1 and 5-methyl cytosine (5MeC) expression in normal and transgenic BF cells in which DNMT-1 is targeted for silencing.

Sample Population – Normal and transgenic BF cells

Procedure – BFs were transfected with a short hairpin RNA (shRNA) designed to silence the expression of DNMT-1, using a lentiviral vector with green fluorescent protein (GFP) as a reporter gene.  In order to evaluate and quantify DNMT-1 expression and function, normal and transgenic BF cells were labeled for DNMT-1 and 5MeC using fluorescent immunocytochemistry (ICC).  Quantitative analysis of DNMT-1 and 5MeC was conducted using NIS Elements Software.

Results – Average intensity measurements from fluorescent label of 5MeC were significantly lower (p<.0001) from BFs that contained approximately 40% transgenic cells when compared to normal BFs.  This suggests that the shRNA did in fact silence the expression of DNMT-1, which in turn reduced the amount of methylation on the DNA.

Conclusions and Clinical Relevance – Transgenic cells had less methylation than normal BF cells which suggests that DNMT-1 was silenced.  The use of transient RNAi to knockdown DNMT-1 expression in cells prior to cloning may be a useful tool that can be applied to correct aberrant reprogramming that occurs in preimplantation cloned embryos.

Impact for Human Medicine – In addition to the important role epigenetics plays in directing normal development, it is also known that abnormal epigenetic control of gene expression results in a variety of human disease conditions including cancer and other developmental abnormalities like Angelman syndrome (Brown R., 2002.)