RNA Interference-mediated Gene Silencing Research Articles

RNA-Induced Silencing Complex-Bound Small Interfering RNA Is a Determinant of RNA Interference-Mediated Gene Silencing in Mice

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjögren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.

The possible role of penaeidin5 from the black tiger shrimp, Penaeus monodon, in protection against viral infection

Penaeidin class 5 (PEN5) has so far only been reported in the Chinese shrimp, Fenneropenaeus chinensis, and the black tiger shrimp, Penaeus monodon. The PEN5 homolog from F. chinensis ( FenchiPEN5) exhibits antimicrobial activities against both Gram-positive and Gram-negative bacteria as well as fungi. Here, we characterized the PEN5 gene from P. monodon ( PenmonPEN5) and evaluated its potential involvement in antiviral immunity. The deduced open reading frame of PenmonPEN5 encodes for a predicted 79 amino acid peptide including a 19 amino acid signal peptide. The gene structure of PenmonPEN5 contains two exons interrupted by one intron, whilst the 5′ upstream sequence contains a putative TATA box and several GATA, GATA-3, AP-1 and dorsal transcription factor binding sites. PenmonPEN5 mRNA levels in P. monodon shrimps following a systemic infection with white spot syndrome virus (WSSV) were significantly induced at 24 h post infection, but was strongly down-regulated at 48 h post injection, compared to those of the uninfected control shrimps. The suppression of PenmonPEN5 transcript levels by RNA interference mediated gene silencing led to an increased susceptibility of shrimps to WSSV infection, suggesting a possible role of PenmonPEN5 in the shrimp's antiviral immunity.

Conserved function of the Krüppel gap gene in the blowfly Lucilia sericata, despite anterior shift of expression

To determine whether expression patterns of segmentation genes found in Drosophila melanogaster can be scaled to pattern larger insects, we studied the expression of the Krüppel (Kr) gene in the blowfly Lucilia sericata. Compared with Drosophila Kr, L. sericata Kr showed an unexpected 10% shift of expression towards the anterior pole. Furthermore, expression domains not found in D. melanogaster were present at the blastoderm stage of L. sericata. To compare Kr activity and function, we employed RNA interference-mediated gene silencing. We found Kr phenotypes in L. sericata comparable with those observed in D. melanogaster, demonstrating that L. sericata Kr functions as a gap gene as it does in Drosophila. Our results show that, despite an anterior shift in expression, Kr function has remained conserved during the evolution of the blowflies.

Granzyme B is expressed in urothelial carcinoma and promotes cancer cell invasion

Granzyme B (GrB) is a serine proteinase known to be expressed by cytotoxic lymphocytes and to induce, in presence of perforin (Pf), apoptosis in target cells. Recently, GrB expression has been shown (often in absence of Pf) in nonlymphoid cells, but its function is not defined. In our study, we investigated GrB and Pf expression in bladder cancer cell lines and in urothelial carcinoma (UC) tissues by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, ELISA, immunofluorescence and immunohistochemistry. We also assessed the function of GrB in UC cells; the in vitro function of GrB was examined by loss-of-function experiments. Our results revealed that GrB is expressed, in absence of Pf, in UC cells. Significant differences were found between GrB expression and both increasing pathological tumor spreading and high-grade vs. low-grade pTa tumors. Notably, GrB in UC tissues was concentrated at the cancer invasion front and was expressed in neoplastic cells undergoing epithelial-mesenchymal transition, a key event in carcinoma invasion. Indeed, GrB-positive cells also expressed Snail, N-cadherin or were negative for E-cadherin. GrB expressed in tumor cell lines was enzymatically active and capable of vitronectin cleavage, implying extracellular matrix (ECM) remodeling by GrB. Inhibition of GrB activity or Stealth RNA interference-mediated GrB gene silencing markedly suppressed bladder cancer cell invasion through matrigel. This data provides the first evidence for a role of GrB in promoting cancer cell invasion. Taken together, our findings suggest that GrB, via ECM degradation, contributes to the establishment of the UC invasive phenotype.

Abstract 2950: Silencing of STAT3 and Src gene by RNA interference enhances the cytotoxic effect of cisplatin in cervical cancer cell lines

Abstract Purpose: Signal transducer and activator of transcription-3 (STAT3) is well known to regulate oncogenesis, angiogenesis and immunosuppression in cancer cells. The Src family consists of nonreceptor tyrosine kinases and participates in cell cycle progression, adhesion, spreading, migration, and differentiation. Activation of STAT3 by constitutively activated Src pathway in cancer cells results in cancer progression. We investigated the cytotoxic effect of cisplatin in cervical cancer cell lines according to the presence of RNA interference-mediated STAT3 and Src gene silencing. Experimental procedures: We prepared two cervical cancer cell lines of C4I and HeLa. First of all, reverse transcription with an RT-PCR kit was performed in order to confirm the increased STAT3 expression in cervical cancer cells. We synthesized STAT3-specific siRNA, using six different sequences of oligonucleotides encoding STAT3 siRNA. After ligation and transformation into bacteria, the positive colonies were selected by antibiotic resistance. HeLa cells were transfected with the plasmids with luciferase reagent according to the established methods. As the same methods, we obtained the HeLa cells transfected with c-Src and v-Src-targeting siRNAs. We checked the reduction rate of each mRNAs by RT-PCR after 48 hours. HeLa and C4I cells transfected with indicated siRNAs, which showed the highest reduction rate at silencing STAT3, c-Src and v-Src, were cultured and mock transfection without any DNA was always included as experimental controls. At 36 hours after starting culture, cisplatin was administered as indicated concentrations (0, 100, 250, 500, 1000 ng/mL). At 96 hours after starting culture, the viability of HeLa and C4I cells was determined by the MTT assay. Results: We confirmed the increased expression of STAT3 in HeLa cell lines. STAT3 mRNAs were reduced very effectively in all cases of STAT3-targeting siRNAs with different 6 sequences to HeLa (80.1%∼93.4%) and the following sequence of oligonucleotides encoding STAT3 siRNA showed the highest reduction rate; 5′-GCGTCCAGTTCACTACTAAAGTCAG-3′ (upper strand) and 5′-CTGACTTTAGTAGTGAACTGGACGC-3′ (lower strand). The reduction rate at silencing c-Src with different 5 sequences siRNAs was high (73.8%∼88.9%). The reduction rate at silencing v-Src with different 5 siRNAs was lower than other two cases (40.5%∼66.6%). The enhancement of cytotoxicity of cisplatin by gene silencing of STAT3 and c-Src, but not of v-Src, was confirmed in HeLa and C4I cells. Conclusion: In this study, we confirmed that STAT3/c-Src targeting siRNAs down-regulated the expression of STAT3 and c-Src and they could enhance the cytotoxic effect of cisplatin in cervical cancer cell lines. We will continue the upgraded study to investigate methods for enhancement of drug susceptibility, including the effect of co-silencing STAT3 and c-Src and their in vivo responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2950.

Small RNA interference-mediated gene silencing of heparanase abolishes the invasion, metastasis and angiogenesis of gastric cancer cells

BackgroundHeparanase facilitates the invasion and metastasis of cancer cells, and is over-expressed in many kinds of malignancies. Our studies indicated that heparanase was frequently expressed in advanced gastric cancers. The aim of this study is to determine whether silencing of heparanase expression can abolish the malignant characteristics of gastric cancer cells.MethodsThree heparanase-specific small interfering RNA (siRNAs) were designed, synthesized, and transfected into cultured gastric cancer cell line SGC-7901. Heparanase expression was measured by RT-PCR, real-time quantitative PCR and Western blot. Cell proliferation was detected by MTT colorimetry and colony formation assay. The in vitro invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells.ResultsTransfection of siRNA against 1496-1514 bp of encoding regions resulted in reduced expression of heparanase, which started at 24 hrs and lasted for 120 hrs post-transfection. The siRNA-mediated silencing of heparanase suppressed the cellular proliferation of SGC-7901 cells. In addition, the in vitro invasion and metastasis of cancer cells were attenuated after knock-down of heparanase. Moreover, transfection of heparanase-specific siRNA attenuated the in vitro angiogenesis of cancer cells in a dose-dependent manner.ConclusionsThese results demonstrated that gene silencing of heparanase can efficiently abolish the proliferation, invasion, metastasis and angiogenesis of human gastric cancer cells in vitro, suggesting that heparanase-specific siRNA is of potential values as a novel therapeutic agent for human gastric cancer.

An Ascovirus-Encoded RNase III Autoregulates Its Expression and Suppresses RNA Interference-Mediated Gene Silencing

RNase III proteins play vital roles in processing of several types of RNA molecules and gene silencing. Recently, it has been discovered that some plant and animal viruses encode RNase III-like proteins as well. Genome sequencing of four virus species belonging to the Ascoviridae family has revealed sequence conservation of an RNase III open reading frame among the viruses. These have not been explored in ascoviruses, and therefore their role in host-virus interaction is unknown. Here, we confirmed expression of Heliothis virescens ascovirus (HvAV-3e) open reading frame 27 (orf27) that encodes an RNase III-like protein after infection and demonstrated dsRNA specific endoribonuclease activity of the encoded protein. Analysis of the expression patterns of orf27 in virus-infected insect cells and a bacterial expression system revealed autoregulation of this protein over time. Moreover, HvAV-3e RNase III was found essential for virus DNA replication and infection using RNA interference (RNAi)-mediated gene silencing. In addition, using green fluorescent protein gene as a marker, we provide evidence that RNase III is involved in the suppression of gene silencing. To our knowledge, this is the first insect virus-encoded RNase III described and shown to suppress host cell RNAi defense mechanism.

Application of somatic embryos to rapid and reliable analysis of soybean seed components by RNA interference-mediated gene silencing

Soybean somatic embryos have attracted attention both as a model of zygotic embryos and as explants for the generation of transgenic plants. β-Conglycinin, which is composed of three subunits (α, α′, and β) that are encoded by a multigene family, is a major seed component of soybean, and accumulates in somatic embryos with their maturation. We subjected the somatic embryos to transformation with vectors encoding double-stranded RNA fragments of various sizes that correspond to the gene for the α′ subunit of β-conglycinin. These DNA fragments were put under the control of the promoter region of the gene for another major seed protein, the A2B1a subunit of glycinin. Transgenic somatic embryos were obtained within 2 months of transformation, and the resulting mature embryos manifested down-regulation of the α′ subunit of β-conglycinin in a manner dependent on the size of the RNAi vector insert. Accumulation of small interfering RNA and depletion of mRNA corresponding to the α′ subunit were indicative of RNAi. The amounts of α and β subunits of β-conglycinin, which share high sequence similarity with the α′ subunit, were also reduced in the mature somatic embryos. Moreover, the abundance of all β-conglycinin subunits was greatly reduced in the seeds of regenerated transgenic plants. Our results indicate that the application of RNAi to somatic embryos is a feasible and rapid option for functional studies of soybean seed components.

RNA interference-mediated URG4 gene silencing diminishes cyclin D1 mRNA expression in HepG2 cells

Up-regulated gene 4 (URG4), stimulated by HBxAg, is a novel gene located on chromosome 7 (7p13). The full-length URG4 clone is 3.607 kb and encodes a polypeptide of 922 amino acids, with a molecular weight of 104 kDa (GeneID: 55665). It promotes cell growth, growth factor-independent survival, and anchorage-independent growth in HepG2 cells, and it accelerates tumor formation in nude mice. Hence, URG4 may be a natural effector of HBxAg and a putative oncogene that contributes to multi-step hepatocarcinogenesis. Cyclin D1 is frequently over-expressed in hepatocellular carcinoma, exhibiting a number of malignant phenotypes. We found that down-regulation of URG4 through RNA interference-mediated silencing suppressed cell proliferation in HepG2 cells. Over-expression of URG4 up-regulated cyclin D1 mRNA expression, whereas RNA interference-mediated URG4 silencing diminished cyclin D1 mRNA expression in HepG2 cells. The data suggest that URG4 may play an important role in the development of hepatocellular carcinoma by partially regulating the expression of cyclin D1 and has potential for use as a therapeutic target for hepatocellular carcinoma.

Light activation as a method of regulating and studying gene expression

Recently, several advances have been made in the activation and deactivation of gene expression using light. These developments are based on the application of small molecule inducers of gene expression, antisense- or RNA interference-mediated gene silencing, and the photochemical control of proteins regulating gene function. The majority of the examples employ a classical 'caging technology', through the chemical installation of a light-removable protecting group on the biological molecule (small molecule, oligonucleotide, or protein) of interest and rendering it inactive. UV light irradiation then removes the caging group and activates the molecule, enabling control over gene activity with high spatial and temporal resolution.

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

We describe efficient polyethylene glycol (PEG)-mediated and Agrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus, Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation of C. passeckerianus. The PEG-mediated transformation of C. passeckerianus protoplasts was successful and generated hygromycin-resistant transformants more efficiently than either phleomycin or carboxin resistance. Agrobacterium-mediated transformation with plasmid pBGgHg containing hph gene under the control of the Agaricus bisporus gpdII promoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for the Agrobacterium-mediated system within the genome. C. passeckerianus actin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression in C. passeckerianus, but only when constructs contained a 5' intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated using gfp as a target gene easily scored in C. passeckerianus. Upon transformation of gfp antisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression in C. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.

Suppressed expression of RETROGRADE-REGULATED MALE STERILITY restores pollen fertility in cytoplasmic male sterile rice plants

Conflict/reconciliation between mitochondria and nuclei in plants is manifested by the fate of pollen (viable or nonviable) in the cytoplasmic male sterility (CMS)/fertility restoration (Rf) system. Through positional cloning, we identified a nuclear candidate gene, RETROGRADE-REGULATED MALE STERILITY (RMS) for Rf17, a fertility restorer gene for Chinese wild rice (CW)-type CMS in rice (Oryza sativa L.). RNA interference-mediated gene silencing of RMS restored fertility to a CMS plant, whereas its overexpression in the fertility restorer line induced pollen abortion. The mRNA expression level of RMS in mature anthers depended on cytoplasmic genotype, suggesting that RMS is a candidate gene to be regulated via retrograde signaling. We found that a reduced-expression allele of the RMS gene restored fertility in haploid pollen, whereas a normal-expression allele caused pollen to die in the CW-type CMS. RMS encodes a mitochondrial protein, 178 aa in length, of unknown function, unlike the majority of other Rf genes cloned thus far, which encode pentatricopeptide repeat proteins. The unique features of RMS provide novel insights into retrograde signaling and CMS.

Growth Hormone Inhibition of Glucose Uptake in Adipocytes Occurs without Affecting GLUT4 Translocation through an Insulin Receptor Substrate-2-Phosphatidylinositol 3-Kinase-dependent Pathway

Growth hormone (GH) pretreatment of 3T3-L1 adipocytes resulted in a concentration- and time-dependent inhibition of insulin-stimulated glucose uptake. Surprisingly, this occurred without significant effect on insulin-stimulated glucose transporter (GLUT) 4 translocation or fusion with the plasma membrane. In parallel, the inhibitory actions of chronic GH pretreatment also impaired insulin-dependent activation of phosphatidylinositol (PI) 3-kinase bound to insulin receptor substrate (IRS)-2 but not to IRS-1. In addition, insulin-stimulated Akt phosphorylation was inhibited by GH pretreatment. In contrast, overexpression of IRS-2 or expression of a constitutively active Akt mutant prevented the GH-induced insulin resistance of glucose uptake. Moreover, small interfering RNA-mediated IRS-2 knockdown also inhibited insulin-stimulated Akt activation and glucose uptake without affecting GLUT4 translocation and plasma membrane fusion. Together, these data support a model in which chronic GH stimulation inhibits insulin-dependent activation of phosphatidylinositol 3-kinase through a specific interaction of phosphatidylinositol 3-kinase bound to IRS-2. This inhibition leads to suppression of Akt activation coupled to glucose transport activity but not translocation or plasma membrane fusion of GLUT4.

Growth Hormone Promotes Axon Growth in the Developing Nervous System

Postnatally, endocrine GH is primarily produced by pituitary somatotrophs. GH is, however, also produced in extrapituitary sites, including tissues of the developing nervous system such as the neural retina. Whereas GH roles in the nervous system are starting to emerge, they are still largely unknown. We show here that GH in the neural retina is mainly present in the axons of retinal ganglion cells (RGCs) in embryonic day (E) 4-12 chick embryos, but it is no longer present at E14-18. This temporal window corresponds to the period of RGC axon growth. GH receptor mRNA was also detected within cells of the E7 RGC layer and GH receptor protein colocalized with GH in RGC axons. The possibility that GH promotes axon growth was thus investigated. Exogenous GH induced a significant increase in axon elongation at 10(-9) and 10(-6) M in E7 RGC culture purified by immunopanning. RNA interference-mediated gene silencing was used to examine whether endogenous GH similarly alters axon outgrowth. The ability of GH small-interfering RNA to knock down GH was first tested using HEK cells on a LacZ-cGH expression plasmid and found to reach 90%. Upon transfection of GH small-interfering RNA to immunopanned RGC culture, a 63% knockdown of endogenous GH was detected and RGC axon length was found to be reduced by 40%. Taken together, these data suggest that GH acts as an autocrine or paracrine signaling molecule to promote axon growth in a developing nervous tissue, the neural retina of chick embryos.

Knockdown of proteins involved in iron metabolism limits tick reproduction and development

Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus, and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1, fer2, and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine.

RIG-I-like antiviral protein in flies

The function of gene expression in the response of drosophila to viral infection is poorly understood. A report now demonstrates that the helicase Dicer-2 controls antiviral gene expression in addition to RNA interference–mediated gene silencing.

RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin

We have previously reported that a cytochrome P450, CYP6BG1, from Plutella xylostella was found to be overexpressed in 4th instars of a permethrin resistant strain and inducible in the susceptible counterpart. The findings suggested potential involvement of CYP6BG1 in permethrin resistance, hence warranted a functional analysis. To assess the functional link of the gene to permethrin resistance, we adopted RNA interference-mediated gene silencing (RNAi) by dsRNA droplet feeding. Here, real time PCR analyses show that oral delivery of dsRNA can efficiently reduce the expression of CYP6BG1. Knockdown of CYP6BG1 transcript was evident in midgut and larval tissues enclosed in carcass. As a consequence of knockdown, a significant reduction in resistance of larvae fed CYP6BG1 dsRNA was observed after 24 and 48 h of exposure to permethrin. In addition, CYP6BG1 dsRNA feeding to larvae led to reduced total P450 activities of microsomal preparations toward model substrates p-nitroanisole and benzyloxyresorufin. These results indicate that the overexpressed CYP6BG1 participate in enhanced metabolism of permethrin, thereby, resistance. The knockdown of a non-overexpressed P450, CYP6BF1v4, from the same resistant P. xylostella strain did not lead to changes in the level of resistance to permethrin, supporting further the specific involvement of CYP6BG1 in the resistance.

Correlation of O6-Methylguanine Methyltransferase (MGMT) Promoter Methylation With Clinical Outcomes in Glioblastoma and Clinical Strategies to Modulate MGMT Activity

Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) remains a significant barrier to the successful treatment of patients with malignant glioma. The relative expression of MGMT in the tumor may determine response to alkylating agents, and epigenetic silencing of the MGMT gene by promoter methylation plays an important role in regulating MGMT expression in gliomas. MGMT promoter methylation is correlated with improved progression-free and overall survival in patients treated with alkylating agents. Strategies to overcome MGMT-mediated chemoresistance are being actively investigated. These include treatment with nontoxic pseudosubstrate inhibitors of MGMT, such as O(6)-benzylguanine, or RNA interference-mediated gene silencing of MGMT. However, systemic application of MGMT inhibitors is limited by an increase in hematologic toxicity. Another strategy is to deplete MGMT activity in tumor tissue using a dose-dense temozolomide schedule. These alternative schedules are well tolerated; however, it remains unclear whether they are more effective than the standard dosing regimen or whether they effectively deplete MGMT activity in tumor tissue. Of note, not all patients with glioblastoma having MGMT promoter methylation respond to alkylating agents, and even those who respond will inevitably experience relapse. Herein we review the data supporting MGMT as a major mechanism of chemotherapy resistance in malignant gliomas and describe ongoing studies that are testing resistance-modulating strategies.

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

BackgroundT 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.ResultsWe 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.CoclusionThe 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.

The LEM Domain Proteins Emerin and LAP2α Are Dispensable for Human Immunodeficiency Virus Type 1 and Murine Leukemia Virus Infections

The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.