ÿþ<HTML><HEAD><TITLE>XI International Meeting on Paracoccidioidomycosis</TITLE><link rel=STYLESHEET type=text/css href=css.css></HEAD><BODY aLink=#ff0000 bgColor=#FFFFFF leftMargin=0 link=#000000 text=#000000 topMargin=0 vLink=#000000 marginheight=0 marginwidth=0><table align=center width=700 cellpadding=0 cellspacing=0><tr><td align=left bgcolor=#cccccc valign=top width=550><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=3><font size=1>XI International Meeting on Paracoccidioidomycosis</font></font></strong><font face=Verdana size=1><b><br></b></font><font face=Verdana, Arial,Helvetica, sans-serif size=1><strong> </strong></font></font></td><td align=right bgcolor=#cccccc valign=top width=150><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=1><font  size=1>Resume:92-5</font></em></font></strong></font></td></tr><tr><td colspan=2><br><br><table align=center width=700><tr><td><b>Poster (Painel)</b><br><table width="100%"><tr><td width="60">92-5</td><td><b>Use of antisense RNA technology for silencing of PbP27 gene in Paracoccidioides brasiliensis</b></td></tr><tr><td valign=top>Authors:</td><td><u>Isaura Torres Gomez </u>  (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Orville Hernandez Ruiz   (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Jose F Munoz   (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Diana Tamayo   (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Ana Maria Garcia   (BCM, CIB - Molecular and Cellular Biology Unit) ; Angela Restrepo   (BCM, CIB - Molecular and Cellular Biology Unit) ; Juan G Mcewen   (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Facultad de Medicina, U de Antioquia, Medellin, Colombia) </td></tr></table><p align=justify><b><font size=2>Abstract</font></b><p align=justify class=tres><font size=2>P27 is an antigenic protein produced by P. brasiliensis<i>text</i>, the etiologic agent of paracoccidioidomycosis (PCM). This protein has been considered as a putative virulence factor, and proposed as a suitable target for the designing diagnostic tools and vaccines. To evaluate the role of this antigen, we designed four antisense oligonucleotides vectors targeting PbP27 and using the transformation system mediated by Agrobacterium tumefaciens<i>text</i> (ATMT), we transformed yeast cells from the strain Pb339. The Hygromycin resistance was used to select the transformed isolates with the antisense molecules as well as the yeast with empty vector (PbWt+EV). The isolates were evaluated at the transcriptional and translational level: (i) PbP27 gene expression was determined by quantitative reverse transcription PCR (qRT-PCR) performed on a CFX 96 System (Bio-Rad) and (ii) protein synthesis was assayed by western immunoblotting using a monoclonal antibody BJ4, previously reported. 
In this study we generated three different P. brasiliensis<i>text</i> isolates that were mitotically stable (PbP27 aRNA), with down regulation in PbP27 gene expression ranging from 70-75% compared to PbWt and PbWt+EV. This phenotype remained stable after 18 months of subculture in appropriate culture media, which indicates the integration of the T-DNA into the P. brasiliensis<i>text</i> genome, also supported by the amplification of the hph gene. In addition, the reduction of p27 protein was demonstrated in western inmunoblotting assays with BJ4 monoclonal antibody in different extracts obtained from controls and PbP27 aRNA isolates. In summary, aRNA is a suitable silencing mechanism for P. brasiliensis. The implementation of this silencing technology to study antigenic proteins such as p27, constitutes an interesting step in the evaluation of their functions and possible roles as virulence factors involved in PCM pathogenesis. Acknowledgement: Colciencias (project No 221340820447), Corporación para Investigaciones Biológicas and Universidad de Antioquia supported this work. The National Doctoral Program of COLCIENCIAS supported Isaura Torres and Orville Hernández. We sincerely thank Dra Beatriz Gómez by her kind assistance in providing the BJ4 monoclonal antibody.
</font></p><br><b>Keyword: </b>&nbsp;Paracoccidioides brasiliensis, PbP27, Gene Silencing, Real time PCR, Agrobacterium tumefaciens</td></tr></table></tr></td></table></body></html>