• flex switch
• fluorescent protein
• in vivo imaging
• leakage
• mosaicism
• polar angle
• recombination

In tumors and in several diseases tissues are characterized by healthy and diseased cells, creating mosaicism. Currently, the mosaicism phenomenon is hard to understand and reproduce in vivo.
A possible strategy is the generation of conditional and inducible mutations by crossing a floxed mouse with a transgenic animal carrying Cre recombinase. This model can be studied in different conditions, to analyze the mechanisms at the base of development, cellular homeostasis, and tumor progression.
At the present days, mosaicism’s studies are affected by the lack of a tool that reproduces the in vivo mosaicism allowing to examine phenomenon in its entirety, without the need to cross many different transgenic animals.
The tool proposed can satisfy these conditions, by allowing the creation and detection of a conditional genetic knock-out mosaicism with Cre recombinase activity.
Cre recombinase realizes a homologous recombination on a DNA sequence flanked by two Lox sites. Depending on the DNA sequence, if it is a target gene or a STOP site situated in the 5’ portion of a gene, the target gene can be repressed or activated respectively.
Importantly, the generation and control of a mosaic allows to understand not only mosaicism, but the cell-autonomous effect of a mutation. Therefore, it is possible to understand, separately, the contribution of environment and genome on the cell fate.
The tool we devised is formed by a plasmid including the genes of two different fluorescent proteins (a GFP and a RFP) assembled in such a way that only one of the two genes is expressed. In its native form, the plasmid, called Beatrix, expressed the RFP but upon homologous recombination mediated by the enzyme Cre, the expression switches to the GFP. Therefore, this sensor signals with a color shift the presence and activity of Cre. When this sensor is transduced in a conditional mutant mouse, the color shift signals the mutation.
Furthermore, the plasmid presents a DNA sequence coding Cre recombinase. Its expression is achieved only after the complete plasmid recombination, inducing the amplification of Cre activity.
In a floxed animal, the induction of mosaicism is possible with a low level of Cre activity that can be regulated spatially and temporally. In our tool, the induction of mosaicism occurs thanks to the inducer, a plasmid that expresses an inducible Cre recombinase. The inducible Cre is fused with Ligand Binding Domains (LBDs) that define its cytoplasmic localization. Following the administration of a chemical drug, such as Tamoxifen (4OHT), the inducible Cre changes in the intracellular localization associated to the nuclear localization. In the nucleus, inducible Cre recombines floxed DNA sequences (among which our sensor) on plasmids and genome.
Indeed, the temporal window of Cre activity can be defined by administering Tamoxifen.
However, the only presence of the receptor domain it is not sufficient to obtain the complete cytoplasmic localization, inducing a background Cre activity without the administration of Tamoxifen (leakage). Moreover, due to the presence of Cre recombinase in our construct, the leakage of inducible Cre is increased, inducing recombination in temporal windows not defined by operators. So, the system is not reliable at 100%.
To solve this problem, the system has been modified with the fusion of a destabilized domain (DD), that induces the protein degradation by proteasome 26S, and the removal of the PolyA, that induces the mRNA destabilization.
In presence of Trimethoprim, DD is stabilized, preventing the protein degradation.
In this thesis are proposed several versions of inducible Cre recombinase to define which one has the smallest leakage, so the optimal version for the tool. We transfected the constructs in different cell cultures (HEK293T, NIH, Mouse fibroblast primary cell) through both chemical (Effectene and Lipofectamine) and physical methods (Electroporation).
The acquisition of the cell cultures was performed by means of two-photon microscopy and the images have been analyzed on the fluorescence with specific software.
After the generation of the optimal system to create a genetic mosaicism in cell cultures, the next step is the in vivo application on floxed animals to reproduce and study genetic inactivated mosaicism on whole organism.