Quantitative analysis of siRNA-mediated GFP silencing in transgenic pine cells

Abstract

A quantitative analysis approach of siRNA-mediated green fluorescence protein gene ( gfp) silencing was established in this investigation. This approach is based on both the application of transgenic cells using GFP as a visual marker and siRNA-mediated gfp silencing in Virginia pine ( Pinus virginiana Mill.). We used m-gfp5-ER as a reporter gene to examine design rules for efficient gene silencing in terms of both the levels of mRNA degradation in transgenic cells showing gfp silencing and the degree of silencing by quantitative green fluorescence analyses of confocal images. Using this method, silencing of the m-gfp5-ER gene in gfp transgenic cells can be efficiently determined. Moreover, it can be used to measure the degree of silencing derived from gfp transgene at different developmental stages and at high efficiency. This investigation may provide a useful alternative to study signaling mechanisms of gene silencing in transgenic cells. The method described here allows the quantitative analysis of gene silencing and may also facilitate function discovery of novel plant genes based on siRNA-mediated gene silencing.