The use of gene-silencing strategies to examine the role of periostin during Wolffian duct morphogenesis

Abstract

Background: The research group of Dr. Hinton's laboratory has been investigating molecular mechanisms involved in the control of Wolffian duct (WD)/epididymis morphogenesis, which in turn is necessary for normal male fertility. Recently, this group has demonstrated the presence of periostin within the extracellular matrix (ECM) surrounding the developing WD and has been hypothesized to regulate elongation and coiling of the WD. Objectives: In this study, it is proposed to: 1-) learn the method of dissection of WD from mice embryos and how to maintain these ducts in the culture; 2-) to knock down the expression of periostin using morpholinos oligonucleotides, siRNA and shRNA and examine the effectiveness of each approach and, 3-) to examine WD/epididymis elongation and coilling following periostin knockdown using the most effective approach discovered in (2). Methods: The WD will be dissected and maintained in culture and periostin antisense morpholino, shRNA plasmids and periostin siRNA will be used with the aim of knocking down the expression of periostin. mRNA levels will be quantified by Real Time-PCR. Quantitative evaluation of WD/epididymal coils/bends will be performed following treatment. The localization of periostin and the expression of mitotic cells will be evaluated by immunofluorescence. Expected results: We expect that, at least, one of the RNAi approaches will lower periostin expression levels up to 90%. In the periostin silenced WD, a decreased number of coils will be expected, suggesting that periostin plays an important role in the ECM malleability, promoting elongation and coiling of WD accordingly. With this new approach in my laboratory, I intend to pursue new research directions in collaboration with Dr. Hinton and attract students and researchers to create a research group in this field and consolidate this new model of study in my Department. (AU)