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. 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

Here, we clearly show, that we are able to subsequently tune gene expression over a wide linear range from almost 0% to 100% as compared to perfect and no binding sites. For detailed measurement procedure descriptions, see methods

Figure 1: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_H1 ...

even better:

Figure 1: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_U6 ...

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.