RESUMO
Plasmids containing four GFP-tagged isoforms of the human GDNF gene, with both pre- and pro-regions (pre-pro- GDNF), with the pre- (pre-GDNF) or the pro-region (pro-GDNF) alone, and without the pre- and pro-regions (mGDNF), were used to transfect HEK293 cells (human embryonic kidney cell line). The effect of the transgenic products on the growth of processes was studied in the spinal ganglia of 14-day rat embryos. Media conditioned by the transgenic cells were used to culture explants and dissociated cells of embryonic dorsal root ganglia attached to the bottom of the plate. Medium conditioned by gfp-transgenic HEK293 cells was used as the control. Spinal ganglia explants and dissociated cells cultured in a medium supplemented with recombinant GDNF (recGDNF) as well as in conditioned media containing the pre-GDNF and mGDNF products demonstrated active growth of processes immunopositive for neuronal marker beta-3-tubulin as early as on culture day 4. The ganglia and cells cultured in control medium and media conditioned by cells transgenic for pro-GDNF had no or very few processes even after 10 days of culture.
Assuntos
Axônios/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Transgenes/genética , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/crescimento & desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Células HEK293 , Humanos , RatosRESUMO
The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.