Construction And Application Of Cre Recombinase-mediated Inducible Neuron-death System

With the aging of the society, the increasing rate of cerebrovascular and neurodegenerative diseases reduces the quality of people’s lives seriously by impairing neuronal function. How to repair the damaged neurons is a key issue for the treatment of related diseases. A lot of pathological models have been developed to simulate the damage caused by the diseases and to explore the possible therapeutic approach. However, a variety of current pathological models not only induce neuronal death, but also cause drastic changes of the microenvironment of the damaged zone. This restricts the regeneration and repair of neural circuits seriously. How to reduce the changes of microenvironment caused by pathological model and maximize the reconstruction of neural circuits is a key problem in the research of developmental neurobiology. Thus, it is extremely necessary to construct a specific neuron-death system with limited effect rather than undermining other cells in the central nervous system and to create a new pathological model which just impair the neurons.Based on the Matsuda’s method of controlled expression of transgenes introduced by in vivo electroporation, we constructed two plasmids:Thy-1-CreERT2-3’UTR and CMV-loxP-TagRFP-SV40polyA-loxP-DTR-2AP-EGFP, and established an applicable method of gene transfection by in vivo electroporation. In Vitro and in Vivo experiments verified that all of the functional elements of the system can achieve the desired results. The Thy-1.2promoter mediated neuron-specific expression system and the diphtheria toxin receptor eukaryotic expression vector have been constructed and detected. The results showed that the Thy-1.2regulatory element amplified from the genome of C57BL/6was capable to mediate the specific expression of exogenous genes in neuron. The diphtheria toxin receptor (DTR) eukaryotic expression vector could drive the transfected cells into diphtheria toxin sensitive. The TagRFP expression elements with loxP sites was capable to express red fluorescence protein. The embryos were successfully transfected by in utero electroporation technique and the embryos’overall survival rate was90%, the positive rate of transfected embryos was80%.The co-transfected mouse cortical neurons would conditionally express DTR after the treatment of tamoxifen and will then be killed by diphtheria toxin specifically. Combining with the in Vivo and in Vitro microscopy imaging technology, this system is expected to investigate the reactions triggered by specific neuron-death and to clarify the regeneration and reconstruction of damaged neural circuits. This would provide new ideas and theoretical foundations for treatment strategies after nerve injury.