INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Regulable adenovectors harboring Yamanaka genes as a novel tool for implementing regenerative therapy in the aging brain
Autor/es:
CHIAVELLINI, PRISCILA; CANATELLI-MALLAT, MARTINA; GOYA, RODOLFO G.; LEHMANN, MARIANNE; MOREL, GUSTAVO R.
Lugar:
Mar del Plata
Reunión:
Congreso; Reunion Anual de Sociedades de Biociencia; 2019
Institución organizadora:
Sociedad Argentina de Investigación Clínica
Resumen:
Biological restoring of aging hallmarks by partial cell reprogramming is an emerging avenue of research. In this context, regulable pluripotency gene expression systems are the most widely used at present. A regulable bidirectional adenovector expressing green fluorescent protein (GFP) and oct4, sox2, klf4 and c-myc genes (OSKM) was constructed. Gene expression is controlled by a Tet-Off system. The promoter is placed between OSKM arranged as a bicistronic tandem (hSTEMCCA tandem) and the GFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. In order to generate the virus, linearized plasmidic DNA was transfected in HEK293 Cre cell line, and subsequently infected with H14 helper adenovector, which provides in trans all the viral genes. Given that H14 has its packaging signal flanked by loxP sites and that Cre recombinase is produced by the cell line, H14 is unable to package its DNA. The newly-generated vector was expanded by 5 iterated co-infections of the above cells and the adenovector purified by ultracentrifugation in a CsCl gradient. Titer was 1.2 x 1012 physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the 4 Yamanaka genes. Three days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced GFP expression. This adenovector constitutes a promising tool for implementing non-integrative partial cell reprogramming.