Team:Heidelberg/Parts/Characterization
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[[Image:Promoter_test_220910_hd2010.jpg| thumb | 700px | centre | Promoter strength characterization of tuning constructs in HEK 293 T-REx cell line]] | [[Image:Promoter_test_220910_hd2010.jpg| thumb | 700px | centre | Promoter strength characterization of tuning constructs in HEK 293 T-REx cell line]] | ||
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===Characterization of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337035 BBa_K337035] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337036 BBa_K337036] in different cell lines=== | ===Characterization of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337035 BBa_K337035] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337036 BBa_K337036] in different cell lines=== |
Revision as of 05:38, 27 October 2010
![](https://static.igem.org/mediawiki/2010/9/92/Bg_plasmid.png)
Characterizationpartsexperimental characterizationsCharacterization of promoters in tuning constructs in T-Rex cellsDual Luciferase Assay Table 1
Characterization of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337035 BBa_K337035] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337036 BBa_K337036] in different cell linesIf transfecting [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337035 BBa_K337035] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337036 BBa_K337036] are transfected into different cell lines it is obvious that Hek293T cells are the easiest to transfect with both constructs an expression of 17-22 RLU is to be measured. Hek T-Rex cells are showing and expression level of 12 RLU of both constructs. Hela cells are also showing constant expression levels of 8 RLU with both constructs. A rather low expression of 2RLU is to be seen by transfecting the 2 constructs in Huh7 cells. This might be due to low transfection efficiency of this cell line in general. All together it is to say that [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337035 BBa_K337035] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K337036 BBa_K337036] show comparable expression.
characterization of part [http://partsregistry.org/Part:BBa_K337036 BBa_K337036]Regulation of gene expression can be achieved by fusing miRNA binding sites right behind a GOI. In case a referring shRNA miR is expressed, the GOI is knocked down. Strength of regulation thereby depends on binding site properties. We are able to tune gene expression linearly over a broad range. This is a first proof of principle for various miRNA-mRNA interaction in vitro. Therefore, we transfected HeLa cells in principle with our [http://partsregistry.org/Part:BBa_K337036 pSMB_miTuner Plasmid HD3]. It turned out, that there was no obvious effect of different binding sites on reporter gene expression (data not shown). We assume that the RSV driving the shRNA miR is too weak for tight regulation of the referring binding site behind the GOI. Only if a sufficient amount of shRNA miR binds to its target, translation is significantly repressed. Thus, we expressed the shRNA miR from a separate plasmid which was always co-transfected with the original tuning construct. The reporter genes - i. e. Luc2 and hRluc - were also expressed from separate plasmids to get a reference as well as a transfection control. Then, we conducted a Dual Luciferase Assay for quantification of gene expression. The data preciously shows a tuned expression from almost 0% to 100% (Fig. 1, Fig. 2). Lowest expression refers to complete knockdown through fusion of perfect binding sites (always green bar on the left hand side of the figures) to the reporter gene. 100% means ordinary expression from a construct without binding sites (always orange column on the right hand side of the figures). In presence of the specific shRNA miR, gene expression was mediated to various levels through interactions with the different imperfect binding sites. Whereas, when an unspecific shRNA miR was expressed, gene expression remained unaffected (see raw data below). The latter aspect reveals, that the binding sites were correctly designed, since they seem to interact specifically with a referring shRNA miR. The constructs were tested in two different backbones: pBS_U6 and pBS_H1. Both are in viral context, meaning that they contain inverted terminal repeats (ITRs). The constructs can be packed into the capsid of an adeno-associated virus (AAV). Those constructs we also chose for virus production to infect cells even more efficiently as compared to transfections. Because of the significant data, we decided to inject the viruses into mice to see the tuning effect also in vivo. The pBS_H1 construct should be preferred for mice injections since the expressed shRNA miR against human alpha-1-antitrypsine (hAAT) is cytotoxic in higher concentrations. The pBS_H1 backbone leads to moderate expression ranges, still obviously showing the tuning effect. Strikingly, the order of constructs in terms of knockdown for the imperfect binding sites is similar. M4, M5 and M6 always show strong knockdown, whereas M9, M10 and M11 show only loose down-regulation. Consulting the binding site sequences, the weak knockdown can be addressed to bulges in the supplementary region or complete lack of the 3' region of the binding site. Still high strength could be maintained due to only single nucleotide exchanges in the central region of the binding site.
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