Knockdown of the Dnmt1s transcript using small interfering RNA in primary murine and bovine fibroblast cells.
RNA interference (RNAi) has rapidly developed into one of the most widely applied technologies in molecular and cellular research, and although young, is now an essential experimental tool. The versatility of RNAi, especially in mammalian species, lends to its potential applications in a wide array of fields. Without having to genetically manipulate the genome, the ability to selectively reduce the level of a specific transcript using small interfering RNA (siRNA) molecules has great appeal in studying reprogramming issues in somatic cell nuclear transfer (SCNT) embryos. In such embryos, the aberrant expression of the somatic isoform of Dnmt1 (Dnmt1s), the enzyme responsible for maintaining DNA methylation in all somatic cells, has been implicated as one factor in the improper reprogramming of the donor genome. In the present study, the ability to develop a method allowing for the knockdown, or reduction, of Dnmt1s in primary fibroblast cells, like those commonly used as karyoplast donors in SCNT studies, was investigated in primary murine and bovine fibroblast cells as well as in a compromised cell line (NIH/3T3). Two Dnmt1s-specific siRNA candidates were designed and tested. Using optimized conditions, these siRNAs were transiently transfected into the cells with total RNA and nuclear protein being collected. A 56.5% knockdown in Dnmt1s was achieved in the compromised and primary murine cells whereas Dnmt1s was reduced by 15.4% in the primary bovine cells. A reduction in Dnmt1s mRNA did not correspond to a reduction in protein as determined by immunodetection of Western blots. Overall, this study demonstrated the ability of siRNA to knockdown Dnmt1s mRNA in primary fibroblast donor cells. In order to substantially increase the efficiency while decreasing the anomalies seen in SCNT, novel techniques, like the one proposed, are needed to assist the oocyte's ability to reprogram a differentiated genome.
Adams
A M
AM
Animal and Dairy Science Department, University of Georgia, 425 River Road, Athens, Georgia 30602, USA.
Pratt
S L
SL
Stice
S L
SL
D000818:Animals; D002417:Cattle; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation; D005347:Fibroblasts; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D015151:Immunoblotting; D051379:Mice; D041681:NIH 3T3 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D034741:RNA, Small Interfering; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse