Team:Heidelberg/Project/Measurement Standard
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==Abstract== | ==Abstract== | ||
- | With the increasing importance of small RNA molecules in gene therapy the characterization of miRNAs and their binding sites become crucial for innovative applications. In order to exploit the miRNA ability to target and regulate specific genes, we constructed a measurement standard not only to characterize existing miRNAs but also to validate potential synthetic miRNAs for a new therapeutic approach. The synthetic miRNAs we created lack endogenous targets and are thus applicable for gene regulation without any side effects. This openes new possiblities of precise expression tuning. | + | With the increasing importance of small RNA molecules in gene therapy the identification and characterization ion of miRNAs and their binding sites become crucial for innovative applications. In order to exploit the miRNA ability to target and regulate specific genes, we constructed a measurement standard not only to characterize existing miRNAs but also to validate potential synthetic shRNA miRNAs for a new therapeutic approach. The synthetic miRNAs we created lack endogenous targets and are thus applicable for gene regulation without any side effects. This openes new possiblities of precise expression tuning. |
- | Our miMeasure plasmid normalizes knockdown of the green fluorescent protein ( | + | Our miMeasure plasmid normalizes knockdown of the green fluorescent protein (EGFP) to the blue fluorescent protein (EBFP2). This allows an accurate study of binding site properties, since both fluorescent proteins are combined in the same construct and driven by the same bidirectional promoter. Another advantage is, that any desired binding site can be cloned easily into the miMeasure plasmid with the BB_2 standard. As the binding site is inserted downstream of EGFP, a regulation of EGFP expression is to be expected. |
- | The percentage of knockdown of each modified binding site can be determined by the | + | The percentage of knockdown of each modified binding site can be determined by the comparison of the ratio of EGFP to EBFP2 ratio compared to the ratio of the perfect binding site caused knock-down. The ratio is derived from a linear regression curve. Therefor the knock-down efficiency can be conducted by various basic methods e.g. plate reading, flow cytometry or microscopy. |
==Introduction== | ==Introduction== | ||
- | Micro RNAs regulate the translation of their target genes by binding to regions in the 3’ UTR | + | Micro RNAs regulate the translation of their target genes by preferably binding to regions in the 3’ UTR which are called miRNA binding sites (BS)(ref). This miRNA BS consists of a bp seed region at the 5'UTR that is perfectly matched to the miRNA, and surrounding regions that matched partially. The seed region is defined as being the minimal required basepairing at the 5’ end of the miRNA that can regulate the mRNA. Apart from the seed region, binding can be unspecific, creating bulges between miRNA and mRNA (fig). The position and properties of the bulges seem to play a role in miRNA binding and therefore knockdown efficiency (reviewed in Bartel et al., 2009). |
Since we were going to use synthetic miRNA BS in our genetherapeutic approach, we had to find a way to study their effects in a standardized manner that would be comparable and reproducible. | Since we were going to use synthetic miRNA BS in our genetherapeutic approach, we had to find a way to study their effects in a standardized manner that would be comparable and reproducible. | ||
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To measure GFP and BFP fluorescence intensity, we used microscopy and flow cytometer. Fluorescence was fist evaluated using the Leica DM IRB epifluorescence microscope. Only cells which were positive for transfection were measured. | To measure GFP and BFP fluorescence intensity, we used microscopy and flow cytometer. Fluorescence was fist evaluated using the Leica DM IRB epifluorescence microscope. Only cells which were positive for transfection were measured. | ||
- | First, the cells were washed with 1x PBS and detached from the plate using Trypsin. 30µl Trypsin was added to each well, incubated for ten minutes at room temperature. Cells were resuspended in 170µl | + | First, the cells were washed with 1x PBS and detached from the plate using Trypsin. 30µl Trypsin was added to each well, incubated for ten minutes at room temperature. Cells were resuspended in 170µl 1%BSA in PBS and replicates for each condition were pooled into 24 well plates. 200µl from each well were used for FACS measurements, 100-150µl were used for confocal microscopy. |
FACS | FACS |
Latest revision as of 01:18, 24 October 2010
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