I am studying some plant promoter activity. For this purpose I used commercial plasmid to which I inserted examined promoter sequence. The sequence was inserted above two fused marker genes: Gus and Egfp. Wounded leaves was transformed by Agrobacterium tumefaciens carrying the construct. Callus was induced, selected by antibiotic and then shoots was regenerated. I found no expression of examined genes (Gus and Egfp) and very high expression of housekeeping gene (actine). Ct for actin was around 11 cycle. At the level of genomic DNA actine rose at 11 cycle but Gus and Egfp at 32 cycle. Why I obtain such poor results for Gus and Egfp? Is it the case of poor transformation efficacy or maybe epigenetic regulation of inroduced promoter and marker genes? What is your experience? What would you suggest?
I would use normal PCR to check for the presence of your insert in your potentially transformed plants. It's very common to have epigenetic silencing of transgenes (as you mentioned), but the integrated DNA will always show up with normal PCR. Just remember to include a full set of controls. Good luck!
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