Gene silencing by RNAi in mouse Sertoli cells
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Gene silencing by RNAi in mouse Sertoli cells Emilio González-González, Pedro P López-Casas and Jesús del Mazo* Address: Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain Email: Emilio González-González - [email protected]; Pedro P López-Casas - [email protected]; Jesús del Mazo* - [email protected] * Corresponding author
Published: 11 July 2008 Reproductive Biology and Endocrinology 2008, 6:29
doi:10.1186/1477-7827-6-29
Received: 11 April 2008 Accepted: 11 July 2008
This article is available from: http://www.rbej.com/content/6/1/29 © 2008 González-González et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background: RNA interference (RNAi) is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium. Methods: The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein). RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs) targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1) was used. Cell types, the efficiency of both transfections and RNAi were all evaluated. Results: Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low. Conclusion: In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.
Background RNA interference (RNAi) describes any process in which double stranded RNA (dsRNA) triggers post-transcriptional gene silencing. Strategies for inducing gene silencing, either for the study of gene function or in a therapeutic context, have been developed [1]. Small interference RNAs (siRNAs) and short hairpin RNAs (shRNAs) have been used in vitro and in vivo for interfering with RNA [2-5]. siRNAs are dsRNAs of 21–23 base pairs (bp) generated by chemical synthesis [6], enzymatic cleavage [7] or expression systems [8], while shRNAs are dsRNA molecules that mimic endogenous pre-micro RNAs (pre-miR-
NAs). shRNAs consist of two palindromic sequences of 19–29 nucle
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