IMPROVED DESIGN OF ANGIOTENSIN II AT2 RIBOPROBES

CORREA M. MILAGROS; ARCE MARÍA E; CIUFFO GLADYS M.; FUENTES, LB

Los Cocos- Cordoba

Congreso; Reunion Anual de la Soc. de Invest. en Neurociencias; 2014

Soc. de Invest. en Neurociencias

Angiotensin II (Ang II), the active component of the renin?angiotensin system, binds and activates two major subtypes of receptors, namely AT1 and AT2. Ang II receptor expression is highly modulated during brain development. The aim of this work was the generation of AT2 receptor riboprobes to study its expression in rat cerebellum. The PCR fragments of AT2 receptors were subcloned in the p-GEM T easy vector. The identity of the subcloned inserts was verified by RFLP. The restriction endonucleases Eco RI, Ssp I and Pvu II were used. The insert was extracted from the vector with restriction enzymes. The AT2 riboprobes were obtained by transcription in vitro using SP6 or T7 RNA polymerases in both sense and antisense orientations to provide non-specific control and specific probes. The AT2 riboprobes were labeled with non-radioactive digoxigenin and analized by Northern blot. In situ hybridization was performed using the synthetized AT2 riboprobes on fresh frozen coronal sections of PND15 rat cerebellum. We observed specific signal in Purkinje cell layer in coincidence with our previous data by autoradiography and immunohistochemistry. In conclusion, the AT2 riboprobes generated here allows sensitive and efficient detection of AT2 receptor gene expression in rat cerebellum.