Team:Heidelberg/Project/miRNA Kit

miTuner - a kit for microRNA based gene expression tuning in mammalian cells

Abstract

… key regulators, one way, how specificity of gene therapy can be approached (beside target cell tropism of aavs)

Introduction

MicroRNAs (miRNAs) are short endogenous, non-coding RNAs that mediate gene expression in a diversity of organisms [http://2010.igem.org/Team:Heidelberg/Project/miRNA_Kit#References (Bartel, 2004)]. Although the understanding of their biological functions is progressing remarkably, the exact mechanisms of regulation are still not unambiguously defined. However, it is commonly believed that miRNAs trigger target mRNA regulation by binding to 3’ untranslated region (UTR) of its target [http://2010.igem.org/Team:Heidelberg/Project/miRNA_Kit#References (Chekulaeva and Filipowicz, 2009)]. Exact principles of expression knockdown mediated by miRNA are still in debate [http://2010.igem.org/Team:Heidelberg/Project/miRNA_Kit#References (Eulalio et al., 2008)].
However, sequence depending binding site properties have an essential impact on miRNA-mRNA interaction. Depending on pairing specificity translational repression is mediated through the imperfect miRNA-mRNA hybrids. The potential for stringent regulation of transgene expression makes the miRNA world a promising area of gene therapy [http://2010.igem.org/Team:Heidelberg/Project/miRNA_Kit#References (Brown et al.,2009)]. There is a need for tight control of gene expression, since cellular processes are sensitive to expression profiles. Non-mediated gene expression can lead to fatal dysfunction of molecular networks. It is widely known, that miRNAs can adjust such fluctuations[http://2010.igem.org/Team:Heidelberg/Project/miRNA_Kit#References (Brenecke et al., 2005)]. A combination of random and rational design of binding sites could become a powerful tool to achieve a narrow range of resulting gene expression knockdown. To ease in silico construction of miRNA binding sites with appropriate characteristics for its target, we wrote a program - the miBS designer. Using all of our theoretical models gives the user the opportunity to calculate knockdown percentages caused by the designed miRNA in the target cell. Our synthetic miRNA Kit guarantees at least for individually modifiable but still ready-to-use constructs to interfere genetic circuits with synthetic or endogenous miRNAs. We preciously show, that gene expression can thereby by adjusted - tuned - to an arbitrary level. The miTuner (see sidebar) allows on the simultaneous expression of a synthetic miRNA and a gene of interest that is fused with a designed binding site for this specific miRNA. Our modular kit comes with different parts that can be combined by choice, e. g. different mammalian promoters and characterized binding sites of specific properties. By choosing a certain binding site to tag the GOI, one can tune the expression of this gene.

evaluation

Depending on the GOI, different means for read out of gene expression come into play. At first, we applied dual-luciferase assay, since we used Luciferase as a reporter for a proof-of-principle approach. Later on, semi-quantitative immunoblots were prepared for testing of therapeutic genes. However, all the received information fed our models, thereby creating an integrative feedback loop between experiments and simulation.

Results

After creating a binding site library and testing the miRNA-binding site interaction in vitro, we were able to compute an in silico model based on a machine learning approach to predict knockdown efficiencies. A more detailed description of the different binding sites, we characterized can be found in our measurements page.

Another application of our synthetic miRNA Kit profits of tissue specific endogenous miRNAs expression. These can be exploited for On and Off-Targeting. On targeting in this case would mean that the presence of a certain miRNA in a cell switches on expression of the GOI. This can be accomplished by using a repressor that is targeted by an endogenously expressed miRNA. We exemplified this scenario by using a Tet Repressor fused with a perfect binding site for miRNA 122, a liver-specific miRNA (REF!). At the same time, the promoter expressing the GOI would be under control of a Tet Operator. Upon presence of the miRNA 122, the Tet Repressor would be knocked down, release the promoter and expression of the GOI could be established. (YOUC AN CHANGE THIS INTO PAST TENSE IF IT WORKED. AND ADD THE OFF SWITCHE; I AM NOT CERTAIN OF WHAT WE DID THERE, AND FIGURES!)

We further tested our kit using a gene that is an interesting candidate for gene therapy, human alpha-1-antitrypsin (haat) (ref, description). In this approach, we tagged haat, that we used as our GOI, with binding sites that we measured and characterized with our miMeasure construct beforehand. This was a first potential therapeutic approach applying ELISA for measurements.

Discussion

Methods

The miTuner was assembled out of different parts. Cloning was done following standard protocols.

Since the miTuner was constructed initially for Dual Luciferase Assay assay, this was the method of choice for tuning quantification.

References

Eulalio, A., Huntzinger, E., and Izaurralde, E. (2008). Getting to the root of miRNA-mediated gene silencing. Cell 132, 9-14. Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297.