Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer
Angelica M. Giraldo A C , John W. Lynn B , Megan N. Purpera A , Todd D. Vaught C , David L. Ayares C , Robert A. Godke A and Kenneth R. Bondioli A D
+ Author Affiliations
- Author Affiliations
A Embryo Biotechnology Laboratory, School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA.
B Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
C Revivicor Inc., Blacksburg, VA 24060, USA.
D Corresponding author. Email: kbondioli@agcenter.lsu.edu
Reproduction, Fertility and Development 21(6) 785-795 https://doi.org/10.1071/RD08233
Submitted: 17 October 2008 Accepted: 3 May 2009 Published: 1 July 2009
Abstract
The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer. DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. The presence of DNMT1 transcript in the donor cell may contribute to perpetuation of the highly methylated status of the somatic nuclei in cloned embryos. The objective of the present study was to determine the methylation pattern of cloned embryos reconstructed with cells treated with DNMT1-specific small interfering RNA (siRNA). Bovine fibroblasts were transfected with a DNMT1-specific siRNA under optimised conditions. The expression patterns of DNMT1 were characterised by Q-PCR using the ΔΔCT method. The level of DNMT1 was successfully decreased in bovine fibroblast cells using a DNMT1-specific siRNA. Additionally, reduction in the expression of DNMT1 mRNA and DNMT1 protein led to a moderate hypomethylation pattern in the siRNA-treated cells. The use of siRNA-treated cells as donor nuclei during nuclear transplantation induced a reduction in methylation levels compared with controls but did not reduce methylation levels to that of IVF embryos. Further studies are required to determine if this level of reduced methylation is sufficient to improve subsequent development.
Additional keywords: DNA methylation, epigenetics, nuclear reprogramming, siRNA.
Acknowledgements
This research was supported by grants from the Louisiana State University Board of Regents through the Board of Regents Support Fund (contract number LEQSF 2005–07-RD-A-01). Approved for publication by the director of the Louisiana Agricultural Experimental Station as manuscript number 2008–230–1907. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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