Target Validation Of Srna With A Gfp Reporter Gene Fusion System

Most regulation of small RNAs (sRNAs) occurs at the posttranscriptional level. We used primer extension, northern blotting, quantitative reverse transcription (RT)-PCR, beta-galactosidase reporter fusion (LacZ), and other techniques to screen candidate targets that interact directly with sRNAs of interest. We used a low-copy-number green fluorescent protein (GFP) reporter gene fusion vector (pXG-10-SF) to determine the direct target genes of specific small RNAs in a simple target gene screening system for Yersinia pestis. Reporter gene fusion systems based on LacZ or GFP are usually used to validate sRNA-mediated target regulation in vivo. A GFP-based reporter fusion method used to identify the sRNA targets in Y. pestis is described here. pXG10-SF is a modified GFP translation vector that contains the PLtetO promoter. GFP is translated from the fusion vector and its fluorescence detected, and the fluorescence intensity reflects the expression of the target gene itself. When the GFP fusion construct is coexpressed with an sRNA of interest, the effects of the sRNA on the regulation of the target gene is reflected in the relative level of GFP fluorescence.