Functional knock-down of human RAD51 for testing the fanconi anemia-BRCA connection

P. Rio, H. Hanenberg

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

One of the proteins essential in homologous recombination is RAD51, a recombinase involved in nucleoprotein filament formation. After DNA damage, RAD51 colocalizes in nuclear foci with other proteins involved in DNA repair. This foci formation is regulated by BRCA2/FANCD1. As Rad51 deficiency is lethal in mice and a human disorder due to defects in RAD51 does not exist, the aim of the current study was to develop a system to knock-down human RAD51 in an inducible form. For this purpose, we took advantage of the regulatable shRNA delivery system developed by Wiznerovicz and Trono [1] based on the cotransduction of target cells with two lentiviral vectors. The first vector carries expression cassettes for the shRNA and for a marker gene. The second vector constitutively expresses the transcription suppressing protein tTRKRAB whose activity can be regulated via doxycycline (DOX). Here, we show the ability of lentiviral vectors expressing shRNAs designed against human RAD51 to downregulate the protein expression in primary human fibroblasts and in HeLa cells. This effect on the protein levels of RAD51 was controllable by DOX and allowed to visualize RAD51 foci formation in cells via a gammaretroviral vector expressing RAD51 fused to the enhanced green fluorescent protein (EGFP). This inducible system for visualization of fluorescence-tagged essential cellular proteins might facilitate to study the role of RAD51 and its interaction with members of the FA/BRCA pathway in response to defined DNA damages.

Original languageEnglish (US)
Title of host publicationFanconi Anemia
Subtitle of host publicationA Paradigmatic Disease for the Understanding of Cancer and Aging
EditorsHolger Hoehn, Detlev Schindler
Pages211-225
Number of pages15
DOIs
StatePublished - Dec 1 2007

Publication series

NameMonographs in Human Genetics
Volume15
ISSN (Print)0077-0876
ISSN (Electronic)1662-3835

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ASJC Scopus subject areas

  • Genetics(clinical)

Cite this

Rio, P., & Hanenberg, H. (2007). Functional knock-down of human RAD51 for testing the fanconi anemia-BRCA connection. In H. Hoehn, & D. Schindler (Eds.), Fanconi Anemia: A Paradigmatic Disease for the Understanding of Cancer and Aging (pp. 211-225). (Monographs in Human Genetics; Vol. 15). https://doi.org/10.1159/000102559