Production of non radioactive riboprobes against the Angiotensin II type 2 receptor

LACREU ML; CIUFFO GM

San Luis

Congreso; Soc de Biologia de Cuyo; 2009

Soc. Biologia de Cuyo

Angiotensin II (Ang II) receptor expression is highly modulated during development, suggesting a role of these receptors in growth and organogenesis. AT2 receptors (AT2R) are abundantly expressed in fetal tissues and thus a potential role for these receptors has been postulated during development, differentiation and certain pathologies. In order to study the expression of AT2R during development, we prepared a riboprobe to detect the rat AT2R mRNA. The starting material was a vector developed previously in the laboratory pGEM –AT2, where a fragment of the coding region was subcloned. The correct sequence and orientation of the cloned fragment were tested by sequencing. Both, antisense and sense riboprobes were generated by in vitro transcription and labelled with biotin. The probes were evaluated on poliacrylamide gels and initially tested by dot blot. The obtained antisense riboprobes were able to sensitively detect AT2R mRNA by northern blot on tissues known to express the receptor. On the other hand the sense RNA probe showed to be adequate as negative control. Thus, we developed a new tool with a great potential to detect the expression level of AT2 receptor.