Development of New EBV-Based Vectors for Stable Expression of Small Interfering RNA to Mimick Human Syndromes: Application to NER Gene Silencing

Abstract

We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells.

Radiation-induced mitotic catastrophe in PARG-deficient cells

“…The BD650 control line expresses a non functional shRNA (Biard et al, 2005). The sequence of the PARG shRNA maps to the catalytic domain of PARG, thus preventing the synthesis of all PARG isoforms, which was confirmed by RT-PCR (Fig.…”

“…siRNA design and cloning into pEBVsiRNA vectors and establishment of stable knockdown and control HeLa clones were carried out as previously described (Biard, 2007;Biard et al, 2005). The RNAi sequence for PARG (NM_003631) stretched nucleotides 2325-2343.…”

“…HeLa cells expressing constitutively H2B-GFP were described elsewhere (Kanda et al, 1998). Establishment of stable PARG knockdown and control H2B-GFP expressing HeLa clones was carried out as previously described (Biard, 2007;Biard et al, 2005), leading to H2B-GFP/PARG KD and H2B-GFP/BD650 cell lines, respectively. All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco/BRL, Invitrogen) supplemented with 10% fetal bovine serum (FBS; AdGenix) and 1% gentamicin (Gibco/BRL, Invitrogen) and 125 μg/ml hygromycin B under 5% CO 2 .…”

Identification of microprocessor-dependent cancer cells allows screening for growth-sustaining micro-RNAs

“…An episomal shRNA system targeting the microprocessor It has been described previously that expressing genetargeting shRNAs from episomal vectors under the control of the H1 promoter is an efficient method of obtaining stable populations of homogenously knockeddown cells (see for instance Biard et al, 2005;Biard, 2007;Le May et al, 2010;Boehler et al, 2011). We constructed a series of such vectors expressing shRNAs targeting either Drosha or DGCR8.…”

“…The H1 promoter-containing Epstein-Barr virus plasmids (pEBP for puromycin selection and pEBH for hygromycin selection) have been described previously (Biard et al, 2005). Individual shRNA or pre-miRNA coding plasmids were constructed by inserting hybridized oligonucleotides (Sigma, Lyon, France) into BglII-HindIII digested vectors.…”