We are engineering a synthetic network in lentiviral vector (i.e. a clock) which will allow expression of mRNA/protein levels with a pre- determined frequency and amplitude, but independently of the cell cycle or other oscillatory endogenous signals in a mammalian cell. The oscillator consists of an activator, which promotes its own transcription, as well as the transcription of a repressor against itself, in agreement with the design of the natural circadian oscillators.
The component of the network are modelled and analyzed mathematically prior to its construction, taking into account how, changing parameters, the strength of the circuit is affected.
As activator we are using the tTA of the TET-system, whose expression is controlled by a CMV_TET promoter responsive to the tTA. The same promoter drives the transcription of a natural microRNA directed against target site placed in the mRNA of the tTA. Repressing tTA at the post-transcriptional level, we will overcome any problem due to the leakiness of the promoter, since the gene will be silenced. So far we have implemented the tet-off system in the original lentiviral vector, and validated its efficiency by experiments of transfection in Hek cells and fluorescence observation, using GFP as readout. Ending up with the implementation of the entire system in the lentiviral vector we are testing it mainly by time-lapse experiments.
One of the novelty of our project consists in packaging the entire synthetic oscillator in a lentiviral  vector, so that we can easily transfer the network in living cells and in an in-vivo animal model. In view of medical application our synthetic circuit will have a significant impact for gene therapy of complex diseases, such as diabetes.
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