The knockdown of Dnmt1 using small inhibitory RNA a method to assist in the reprogramming of a donor genome during nuclear transfer /

by Adams, Allison M.

Increasing evidence has implicated the incomplete or aberrant reprogramming of donor nuclei as a contributing factor to the observed inefficiencies and outcomes inherent with the current technique of nuclear transfer (NT). The reprogramming of DNA methylation patterns is one of many events essential to convert a differentiated cell back into a totipotent cell using the donor eggs’ ooplasm. DNA methyltransferase I (Dnmt1) is the enzyme responsible for maintaining methylation patterns. The somatic isoform of Dnmt1 has been shown to be aberrantly expressed in NT-derived embryos and is implicated in the improper reprogramming of the donor genome. Short inhibitory RNA (siRNA) is capable of post-transcriptionally depleting a cell of a specific gene transcript. Using Dnmt1-specific siRNA, the ability to reduce the supply of Dnmt1 transcripts was tested in murine and bovine primary cells. Results indicate the expression of Dnmt1 was successfully reduced in both cell types.