RNA interference-mediated gene silencing in murine T cells: in vitro and in vivo validation of proinflammatory target genes

BACKGROUND: T cells play a central role in many inflammatory diseases, hence the identification and validation of T cell-specific target genes will increase the understanding of T cell function in pathologic inflammatory situations. RNA interference (RNAi), with its ability to induce specific gene silencing in mammalian cells, represents a powerful technology to investigate and validate the function of pharmaceutical target genes in vitro and in vivo. The aim of the present study was to systematically explore RNAi-mediated gene-silencing of known T cell-specific model signaling molecules in primary murine T cells in vitro and in vivo. RESULTS: We demonstrate that siRNA delivery and subsequent silencing of T cell specific genes is substantially increased, if murine T cells were activated prior siRNA transfection. Silencing of ZAP70, p56Lck as well as PLC-γ1 protein expression resulted in impaired function of T cells in vitro. Furthermore, delayed type hypersensitivity (DTH) was ameliorated in vivo after adoptive transfer of ZAP70-silenced T cells. COCLUSION: The combination of RNAi-mediated gene silencing and adoptive transfer of gene-silenced T cells, thus, may allow the identification and analysis of T cell-specific targets for therapeutic intervention. Additionally, this model system may represent an alternative to conventional time consuming and cost intensive gene targeting approaches

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-1

stealth™ siRNAs (S#1, S#2, and S#3) by using nucleofection. Cell lysates were prepared 24 and 48 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control cells were mock transfected ("-", no siRNA added), or transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression in transfected T cells determined 24 h after nucleofection as fold expression of the house keeping gene β-actin. (B) Western blot analysis of transfected T cells 48 h after nucleofection with siRNAs indicated in (A). Protein knockdown of ZAP70 was detected by ZAP70-specific antibody. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-2

nucleofection. Cell lysates were prepared 24 and 48 h after transfection and mRNA and protein expression were analyzed by RT-PCR (A) and Western blot analysis (B). As control, cells were transfected with a control siRNA pool (Ctrl). (A) RT-PCR results of transfected T cells determined 24 h after nucleofection with Control ("siCtrl"), ZAP70 ("siZAP70"), PLC-γ ("siPLC-γ1") and Lck ("siLck") specific siRNAs. (B) Western blot analysis of transfected T cells 48 h after nucleofection. Protein knockdown of ZAP70, PLC-γ and Lck was detected by specific antibodies. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-5

stealth™ siRNAs (S#1, S#2, and S#3) by using nucleofection. Cell lysates were prepared 24 and 48 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control cells were mock transfected ("-", no siRNA added), or transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression in transfected T cells determined 24 h after nucleofection as fold expression of the house keeping gene β-actin. (B) Western blot analysis of transfected T cells 48 h after nucleofection with siRNAs indicated in (A). Protein knockdown of ZAP70 was detected by ZAP70-specific antibody. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of two independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes-0

Sfected with 0.9 μM ZAP70-specific siRNA pool by nucleofection. Cell lysates were prepared 24, 48 and 72 h after transfection and ZAP70 expression was analyzed by RT-PCR (A) and Western blot analysis (B). As control, cells were mock ("-", no siRNA added) transfected () or cells were transfected with a control siRNA pool (Ctrl). (A) RT-PCR of ZAP70 mRNA expression determined as fold expression of the house keeping gene β-actin. (B) Western blot analysis of ZAP70 was detected by ZAP70-specific antibody. β-actin was used as loading control. Shown are the mean values of triplicates of a representative experiments of four independent experiments.<p><b>Copyright information:</b></p><p>Taken from "RNA interference-mediated gene silencing in murine T cells: and validation of proinflammatory target genes"</p><p>http://www.biosignaling.com/content/6/1/3</p><p>Cell communication and signaling : CCS 2008;6():3-3.</p><p>Published online 6 Aug 2008</p><p>PMCID:PMC2517589.</p><p></p

Nucleic Acid-Sensing Toll-like Receptors Are Essential for the Control of Endogenous Retrovirus Viremia and ERV-Induced Tumors

SummaryThe genome of vertebrates contains endogenous retroviruses (ERVs) that are largely nonfunctional relicts of ancestral germline infection by exogenous retroviruses. However, in some mouse strains ERVs are actively involved in disease. Here we report that nucleic acid-recognizing Toll-like receptors 3, 7, and 9 (TLR 3, TLR7, and TLR9) are essential for the control of ERVs. Loss of TLR7 function caused spontaneous retroviral viremia that coincided with the absence of ERV-specific antibodies. Importantly, additional TLR3 and TLR9 deficiency led to acute T cell lymphoblastic leukemia, underscoring a prominent role for TLR3 and TLR9 in surveillance of ERV-induced tumors. Experimental ERV infection induced a TLR3-, TLR7-, and TLR9-dependent group of “acute-phase” genes previously described in HIV and SIV infections. Our study suggests that in addition to their role in innate immunity against exogenous pathogens, nucleic acid-recognizing TLRs contribute to the immune control of activated ERVs and ERV-induced tumors