Receiving Of Adenoviral Vector For The Study Functions Of Synaptopodin, The Protein Of Spine Apparatus

Synaptopodin is the founding member of a novel class of proline-rich actin-associated proteins highly expressed in telencephalic dendrites and renal podocytes. That protein expresses in dendrites of mature neurons in telencephalon. Synaptopodin exists in 3 isoforms: neuronal Synpo-short (685 AA), renal Synpo-long (903 AA), and Synpo-T (181 AA). All 3 isoforms specifically interact with alpha-actinin and elongate alpha-actinin-induced actin filaments.  According data from recent studies, we can suggest that dendritic spines containing sinaptopodin greatly differ in structural and functional properties from the neighboring spines that do not contain sinaptopodin. Clusters of synaptopodin in spines colocalize with internal functional flow of calcium. Thus, sinaptopodin plays a role in synaptic plasticity, as well as the investigation of its function may provide information related to the understanding of mechanical functioning of synapses. Our aim is to develop strategies and to receive an adenoviral vector. To achieve this goal the following have to be done: to choose the template and PCR conditions, to construct a plasmid with the sequence of the protein sinaptopodin, the supersinapsin promoter and the fluorescent protein mKate2 for co-transfection, to receive an adenoviral vector, to define its titer and biological activity. The main methods of this study were: polymerase chain reaction and plasmid cloning. A commercial kit of reagents «Phusion High-Fidelity PCR Kit» and thermocycler «Applied Biosystems» were used for PCR.  For construction plasmid we used enzymes «New England BioLabs», competent DH5a cells, the plasmid purification kit «QIAGEN DNA» and the software «VectorNTI». We defined the primers system (Synpo-HindIII-fw, Synpo-MluI-rev) and the optimum annealing temperature of primers - 65̊C. The template was cDNA isolated from mouse cortex. We obtained the sequence of spine apparatus protein synaptopodin, which was confirmed by electrophoresis. The system of restriction enzymes for insertion of the nucleotide sequence of synaptopodin and supersinapsin promoter in a plasmid with a fluorescent protein mKate2 was chosen. We purified plasmids after transformation with the product of ligation reaction.In the following we would plan to co-transfect this plasmid, to accumulate an adenovirus vectors in HEK cells, to purify vectors with ultracentrifugation and to define its titer and biological activity.