Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells.
DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through their influence on gene expression. Manipulation of the DNA methylation marks of differentiated cells will allow a better understanding of the different molecular processes associated with chromatin structure and gene expression. The objective of this study was to identify small interfering RNAs (siRNAs) with the ability to reduce DNA methyltransferase 1 (Dnmt1) mRNA and consequently decrease Dnmt1 protein as well as DNA methylation in porcine cells. Fibroblasts from four porcine fetuses were established and cultured in 5% CO2 in air at 38 degrees C. Optimal transfection conditions were evaluated using a FITC-labeled control siRNA. Four Dnmt1-specific siRNAs were evaluated upon transfection of each cell line. A nonsilencing siRNA was used as a negative control. The expression patterns of Dnmt1 were analyzed by Q-PCR. The combination of 1 microg of siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Downregulation of Dnmt1 varied between siRNAs. Transfection of porcine cells with highly effective siRNAs resulted in a drastic reduction of Dnmt1 mRNA and a slight decrease in protein production. However, this small reduction in the protein concentration induced significant genomic hypomethylation. These data suggest that although Dnmt1 mRNA abundance plays an important role during protein regulation, Dnmt1 enzyme is mainly posttranscriptionally regulated. Subsequent use of these cells for cloning, differentiation, and cancer studies will provide insight as to how methylation of the DNA affects genomic reprogramming.
Giraldo
Angelica M
AM
Revivicor, Inc., Blacksburg, VA 24060, USA. agiraldo@revivicor.com
Vaught
Todd D
TD
Fu
Limin
L
Duncan
Alison J
AJ
Vance
Amy M
AM
Mendicino
Michael
M
Ayares
David L
DL
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D000090205:DNA Methyltransferase 3A; D015536:Down-Regulation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D034421:Sus scrofa; D014162:Transfection; D000097584:DNA Methyltransferase 3B
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000097584:DNA Methyltransferase 3B