Part:BBa_K337053
synthetic binding site of shRNA miRhaat (imperfect) KD:69 %%
imperfect binding site of shRNA miRhaat in the tuning construct leads to a knockdown percentage of 69% percent if introduced into the 3'UTR of the gene of interest (GOI)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
characterization
Regulation of gene expression can be achieved by fusing miRNA binding sites into the 3'UTR of a GOI. In case a referring shRNA miR is endogenously present or co-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. 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. This was achieved by co-transfection the tuning construct with corresponding shRNA miRhaat and as a control a miRNA w/o binding sites in the target 3'UTR. The experiment was done in a 96-well plate by plating 5000 Hela cells/well 24h before transfection. Transfection was done using Fugene transfection reagent. 2.5ng of tuning construct were co-transfected with the shRNA miR construct of a concentration of 25 ng (1:10). Then, we conducted a [http://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay 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 cloning of perfect binding sites into the 3'UTR to the reporter gene(always green bar on the left hand side of the figures). 100% refers to 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). We chose the data obtained by the construct with the U6 promoter as this promoter is more efficient than the H1 promoter, ensuring that the system is saturated and ensuring that the data is reproducible. The same constructs were also used for [http://2010.igem.org/Team:Heidelberg/Notebook/Methods#Virus_Production 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 [http://2010.igem.org/Team:Heidelberg/Project/Mouse_Infection in vivo]. The pBS_H1 construct should be used 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. point mut 10 (1), point mut 10 (2) and point mut 11 (2) always show strong knockdown, whereas bulge 16-18, only seed and bulge 9-12(2) 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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