Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer.
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 DeltaDeltaC(T) 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.
Giraldo
Angelica M
AM
Embryo Biotechnology Laboratory, School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA.
Lynn
John W
JW
Purpera
Megan N
MN
Vaught
Todd D
TD
Ayares
David L
DL
Godke
Robert A
RA
Bondioli
Kenneth R
KR
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D015536:Down-Regulation; D046149:Embryo Culture Techniques; D047108:Embryonic Development; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000201:enzymology*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D053652:Nuclear Transfer Techniques; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases