Poster Presenter

Influence Of Neurotrophic Factors Transfected Into Insect Cells On The Development Of Mammalian Neural Tissue
Galina Pavlova, N. Kanaikina and A. Revishchin
Russia

We investigated the possibility to direct the behaviour of mammalian neural stem/progenitor cells by means of co-culturing them with the embryonic tissue expressing human neurotrophic factors (NGF, BDNF or GDNF). We established an in vitro co-culture system containing dorsal root ganglion (DRG) of newborn rat and the embryonic nerve cells carrying human neurotrophic genes, that were located 0.2-0.5 mm apart of DRG. All types of locally implanted cells could direct the growth of the rat DRG axons. Their outgrowth directed toward the transfected cells began after 1-2 h, while in control experiments it was started on the third or forth days. These findings suggest that co- cultures of rat DRG and genetically modified cells present a powerful tool to study the survival and integration of the neuronal population.

In another series of experiments, we analyzed the survival of the hyperexpression of neurotrophic factors prevents the glial scar formation after xenotransplantation. Human embryonic kidney cells (cell line HEK293) were transfected with vectors designed to express Glial-cell-line-derived neurotrophic factor (GDNF). The cells were then transplanted into the caudatum-putamen of CBA mice. Transplanted cells survived in recipient brain as long as for 18 days. Immunohistochemical analysis of brain sections stained for scar (actin, fibronectin, and collagen type IV) showed that transgenic GDNF decrease the glial posttraumatic reaction of recipient brain tissue surrounding the transplanted cells. The results confirm the possibility of usage of GDNF as neuroprotective transgene in cell therapy.

However, to improve the results of gene and cell therapy, it is necessary to have the gene constructs with controllable expression of transgene product. The construct developed in this laboratory contains the promoter of heat shock protein 70kDa (HSP70) of Drosophila. This construct containing reporter gene of blue fluorescent protein (BFP) under hsp70 promoter was introduced into genome of mouse emb ryo stem (ES) cells by means of electroporation. Tranfected ES cells did not show BFP fluorescence in usual culture conditions. However, the fluorescence appeared after the cells were heated up to 39oC for 30 min. Both strategies of gene activation can be used in gene therapy experiments.