Luciferase mRNA Research Articles

Differential Inhibition of mRNA Degradation Pathways by Novel Cap Analogs

mRNA degradation predominantly proceeds through two alternative routes: the 5'-->3' pathway, which requires deadenylation followed by decapping and 5'-->3' hydrolysis; and the 3'-->5' pathway, which involves deadenylation followed by 3'-->5' hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp(3)G, m(7)Gp(3)G, or m(2)(7,3'-O) Gp(3)G) and 3' termini (A(31),A(60), or G(16)) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5' terminus to bind eIF4E, not merely the presence of a blocking group at the 5'-end. Introducing a stem-loop in the 5'-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5'-->3' versus 3'-->5' pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the alpha- and beta-phosphate moieties, m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m(2)(7,3'-O)Gpp(CH2)pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m(2)(7,3'-O)Gpp(CH2)pG, but not m(2)(7,3'-O)Gp(CH2)ppG, were more stable in vivo, indicating that the 5'-->3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m(2)(7,3'-O)Gpp(CH2)pG and 3'-terminal poly(G) had the greatest stability of all mRNAs tested.

PTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruzi

BackgroundTrypanosoma cruzi is a protozoan pathogen of major medical importance in Latin America. It is also an early diverging eukaryote that displays many unusual biochemical features. The completion of the T. cruzi genome project has highlighted the need to extend the range of techniques available to study gene function. To this end we report the development of a stable tetracycline-dependent expression vector applicable to this parasite and describe in detail the parameters of the system.ResultsWe first produced T. cruzi cell lines that constitutively expressed bacteriophage T7 RNA polymerase and the tetracycline repressor protein from a multicopy episome. An integrative vector with an inducible expression site under the control of a tetracycline-regulatable T7 promoter (pTcINDEX) was targeted to the transcriptionally silent ribosomal RNA spacer region of these parasites and transformants selected using a T7 RNA polymerase-dependent hygromycin resistance gene. To test the system we used two marker proteins, luciferase and red fluorescent protein (RFP), and an endogenous parasite protein (a mitochondrial superoxide dismutase). In each case we found that induction was both time and dose-dependent. Luciferase mRNA could be induced by at least 100-fold, and luciferase activity up to 60-fold, within 24 hours of the addition of tetracycline. When we examined RFP induction by confocal microscopy and fluorescence activated cell sorter, we observed very high levels of expression (>1000-fold increase in fluorescence intensity), although this was not synchronous throughout clonal populations. Induction of superoxide dismutase resulted in an 18-fold increase in cellular activity. The observation that a tagged version of the enzyme was correctly targeted to the mitochondrion demonstrates that our expression system may also provide a high-throughput strategy for subcellular localisation.ConclusionOur results show that pTcINDEX represents a valuable addition to the genetic tools available for T. cruzi. The vector system is sufficiently flexible that it should have widespread uses including inducible expression of tagged proteins, generation of conditional knockout cell lines and the application of dominant-negative approaches.

1078. Silencing of Transgene Expression Following Plasmid Based Delivery to Murine Skeletal Muscle Is Dose Dependent

Plasmid based gene delivery to skeletal muscle is an attractive method for the expression of therapeutic proteins. However, the level of transgene expression decreases over time and may be an obstacle to achieving sustained therapeutic levels of proteins. To investigate the mechanism responsible for loss of expression over time, a standard plasmid backbone containing the luciferase gene driven by the CMV promoter was injected into the skeletal muscle of the hind limb of C57Bl/6 mice at either a high dose (125|[mu]|g) or a low dose (25|[mu]|g) followed by electroporation. Transgene expression was monitored over time using in situ CCCD imaging. At the end of the study the quantity of transgene DNA and luciferase mRNA in the muscle was measured by quantitative PCR. At early time points (day 3 and day 7) luciferase activity in the high dose group was 2.5- fold higher than in the low dose group. However, luciferase activity in mice that received the high dose fell 230-fold between day 7 and day 42, while in mice that received the low dose of the same plasmid it fell only 4-fold. Vector DNA levels in the injected muscles at the end of the study (day 84) were not significantly different between the two doses indicating that the 27-fold lower luciferase activity in the high dose group compared to the low dose group at day 84 was not due to differences in vector DNA levels but rather to a difference in gene expression. Indeed, the luciferase mRNA to vector DNA ratio was 9-fold higher in the low dose group than in the high dose group at this time point. In the same study, a plasmid containing the identical CMV-luciferase expression cassette flanked by matrix attachment sites (MARs) and placed into a plasmid backbone from which all CpG dinucleotides had been removed, was tested at the same doses. At the low dose the CpG-depleted/MARs plasmid exhibited a similar luciferase expression profile to that of the standard plasmid at the same dose. However, in the high dose group the CpG- depleted/MARs plasmid partially prevented the decrease in luciferase expression over time that was observed with the standard plasmid at the high dose. At 28, 42 and 84 days after plasmid injection the CpG- depleted/MAR plasmid expressed 10-, 13- and 9-fold higher luciferase activity respectively than the standard plasmid. Vector DNA levels in the muscle at day 84 were only 2-fold higher in the CpG-depleted/ MAR group than in the mice that received the standard plasmid. These results demonstrate that reducing the plasmid dose can result in a significant improvement in the duration of transgene expression, and that this effect was not due only to improved persistence of the plasmid DNA in the muscle. In addition, use of a CpG-free plasmid backbone plus MARs partially prevented the decline of transgene expression that occurred at the high plasmid dose, suggesting that the presence of CpG's and/or absence of MARs may contribute to loss of expression over time.

Expression of RUNX2 isoforms: Involvement of cap-dependent and cap-independent mechanisms of translation

RUNX2, a major regulator of skeletogenesis, is expressed as type-I and type-II isoforms. Whereas most eukaryotic mRNAs are translated by the cap-dependent scanning mechanism, translation of many mRNAs including type-I and type-II RUNX2 mRNAs has been reported to be initiated by a cap independent internal ribosomal entry site (IRES). Since the dicistronic plasmid assay used to demonstrate IRES has been questioned, we investigated the presence of IRES in RUNX2 mRNAs using dicistronic plasmid and mRNA assays. Our results show that the dicistronic plasmid assay cannot be used to demonstrate IRES in RUNX2 mRNAs because the intercistronic region of dicistronic plasmids containing the 5'-UTRs of both RUNX2 mRNAs operates as a cryptic promoter. In dicistronic mRNA transfection studies the 5'-UTRs of both RUNX2 mRNAs exhibited no IRES activity. When transfected into osteoblastic cells, monocistronic reporter mRNA preceded by the 5'-UTR of type-II RUNX2 (Type-II-FLuc-A100) was translated to a high degree only in the presence of a functional cap (m(7)GpppG); in contrast, luciferase mRNA preceded by the 5'-UTR of type-I RUNX2 mRNA (Type-I-FLuc-A100) was translated poorly in the presence of either m(7)GpppG or a nonfunctional cap (ApppG). Notably, in transfected cells inhibitors of cap-dependent translation suppressed the translation of m(7)GpppG-capped Type-II-FLuc-A100, but not ApppG-capped reporter mRNA preceded by the IRES-containing hepatitis C virus (HCV) 5'-UTR. Our study demonstrates that type-II RUNX2 mRNA is translated by the cap-dependent mechanism. Although efficient translation of type-I RUNX2 mRNA appears to require a process other than cap-dependent, the mechanism of type-I RUNX2 mRNA translation remains to be resolved.

1068. Plasmid DNA Sequences Present in Conventional HSV Amplicon Vectors Cause and Rapid Transgene Silencing by Inactive Chromatin Formation

The HSV-based amplicon vector, a bacteria-virus-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle amplicon). Although the same dose of the minicircle (MC) amplicon vector in normal human fibroblasts resulted in expression of luciferase approximately 20 times higher than that produced by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Similar results were obtained with nondividing normal human astrocytes in culture, precluding the possibility that this phenomenon is fibroblast specific. Finally, nude mice injected with MC amplicon exhibited higher and more sustained expression of luciferase than those injected with conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. These findings are a significant first step towards developing a novel herpes simplex virus (HSV) amplicon vector that can achieve enhanced long-term transgene expression.

MRNA transfection of dendritic cells: Synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level

The occurrence of translation mechanism in the cytosol offers advantages to mRNA transfer over DNA-based transfection in non-dividing cells. Here, we sought to optimize mRNA constructs allowing a high level of protein upon lipofection. We found that luciferase into mouse dendritic cells (JAWSII cells) was ∼20-fold higher when the luciferase mRNA was capped with 3′- O-methyl-m7 5′Gppp 5′G (anti-reverse cap analogue; ARCA) than with m7 5′Gppp 5′G (CAP). Adding a Poly(A) tail of 100 instead of 64 adenosines in cis increased by ∼35-fold more the protein level. Finally, ARCA-Luc-mRNA-A100 construct was 700-fold better efficient than the CAP-Luc-mRNA-A64 one. Moreover, co-transfection with free Poly(A) chains in trans enhanced by 100% the luciferase level. The efficiency of ARCA-mRNA-A100 construct was validated in immature and mature human CD34-derived dendritic cells. Such mRNA construct was also successful to obtain high level of MART-1 tumor antigen.

Mammalian Smaug Is a Translational Repressor That Forms Cytoplasmic Foci Similar to Stress Granules

Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile alpha motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons.

MMTV accessory factor Naf affects cellular gene expression

Mouse mammary tumour virus (MMTV) encodes a viral superantigen (Sag) and a negative acting factor (Naf) which share parts of their coding sequence. Using 2-dimensional gel electrophoresis (2D-DIGE), we could show that at least 10 different cellular proteins were differentially expressed in Naf positive cells. Also, luciferase reporter expression was down-regulated in Naf expressing cells independent of the promoter used and further experiments suggested that this effect was due in part to a decrease in cellular growth rates. Although in Naf positive cells expression of the major sag containing transcript was strongly induced by the synthetic glucocorticoid dexamethasone, the hormone analogue neither influenced luciferase expression nor mRNA expression of selected cellular proteins identified by 2D-DIGE. Taken together, these data support the previous finding that Naf and Sag have separable activities and suggest that Naf may play a role in modulating host cell gene expression during MMTV infection.

Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging

Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.

Interaction of RNA-binding Proteins HuR and AUF1 with the Human ATF3 mRNA 3′-Untranslated Region Regulates Its Amino Acid Limitation-induced Stabilization

ATF3 expression is induced in cells exposed to a variety of stress conditions, including nutrient limitation. Here we demonstrated that the mechanism by which the ATF3 mRNA content is increased following amino acid limitation of human HepG2 hepatoma cells is mRNA stabilization. Analysis of ATF3 mRNA turnover revealed that the half-life was increased from about 1 h in control cells to greater than 8 h in the histidine-deprived state, demonstrating mRNA stabilization in response to nutrient deprivation. Treatment of HepG2 cells with thapsigargin, which causes endoplasmic reticulum stress, also increased the half-life of ATF3 mRNA. HuR is an RNA-binding protein that regulates both the stability and cytoplasmic/nuclear localization of mRNA species containing AU-rich elements. Another RNA-binding protein, AUF1, regulates target mRNA molecules by enhancing their decay. Amino acid limitation caused a slightly elevated mRNA level for HuR and AUF1 mRNA. The nuclear HuR protein content was unchanged, and AUF1 protein increased slightly after amino acid limitation, whereas the cytoplasmic levels of both HuR and AUF1 protein increased. Immunoprecipitation of HuR-RNA complexes followed by reverse transcriptase-PCR analysis showed that HuR interacted with ATF3 mRNA in vivo and that this interaction increased following amino acid limitation. In contrast, the interaction of AUF1 with the ATF3 mRNA is decreased in histidine-deprived cells relative to control cells. Suppression of HuR expression by RNA interference partially blocked the accumulation of ATF3 mRNA following amino acid deprivation. The results demonstrated that coordinated regulation of mRNA stability by HuR and AUF1 proteins contributes to the observed increase in ATF3 expression following amino acid limitation.

The cyclin-dependent kinase 11 interacts with NOT2

The caspase-processed cyclin-dependent kinase 11 (formerly known as PITSLRE) is implicated in apoptotic signaling. However, the mechanism of apoptotic signal transduction through CDK11 p46 is still unclear. We used a yeast two-hybrid screening strategy and identified NOT2 as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11 p46). We demonstrate that CDK11 p46 directly interacts with NOT2 in vitro and in human cells. The NOT domain in the C-terminal part of NOT2 is responsible for the association between CDK11 p46 and NOT2. Both NOT2 and CDK11 p46 predominantly co-localized in the nucleus. Furthermore, we show that overexpression of NOT2 reduces luciferase mRNA and induces apoptosis. However, NOT2 is not phosphorylated by CDK11 p46. These findings suggest that CDK11 may contribute to apoptosis by regulating the activity of NOT2 independent of its kinase activity.

Identification and characterization of upstream open reading frames (uORF) in the 5′ untranslated regions (UTR) of genes in Saccharomyces cerevisiae

We have taken advantage of recently sequenced hemiascomycete fungal genomes to computationally identify additional genes potentially regulated by upstream open reading frames (uORFs). Our approach is based on the observation that the structure, including the uORFs, of the post-transcriptionally uORF regulated Saccharomyces cerevisiae genes GCN4 and CPA1 is conserved in related species. Thirty-eight candidate genes for which uORFs were found in multiple species were identified and tested. We determined by 5' RACE that 15 of these 38 genes are transcribed. Most of these 15 genes have only a single uORF in their 5' UTR, and the length of these uORFs range from 3 to 24 codons. We cloned seven full-length UTR sequences into a luciferase (LUC) reporter system. Luciferase activity and mRNA level were compared between the wild-type UTR construct and a construct where the uORF start codon was mutated. The translational efficiency index (TEI) of each construct was calculated to test the possible regulatory function on translational level. We hypothesize that uORFs in the UTR of RPC11, TPK1, FOL1, WSC3, and MKK1 may have translational regulatory roles while uORFs in the 5' UTR of ECM7 and IMD4 have little effect on translation under the conditions tested.

Translational Regulation of MOS Messenger RNA in Pig Oocytes1

The temporal and spatial translation control of stored mRNA in oocytes is regulated by elements in their 3'-untranslated region (3'-UTR). The MOS 3'-UTR in pig oocytes is both heterogeneous (180, 480, or 530 nucleotides), and it contains multiple U-rich elements and extensive A-rich sequences (CA13CA5CA5CA6). We have examined the role of these potential regulatory elements by fusing wild-type or mutant MOS 3'-UTRs to luciferase mRNA and then injecting these chimeric transcripts into oocytes. We draw six main conclusions. First, the length of the MOS 3'-UTR tightly controls the level of translation of luciferase during oocyte maturation. Second, two U-rich (U5A) elements and the hexanucleotide signal (AAUAAA) are required for translation. Third, mutations, duplications, or relocations of the A-rich sequence reduce or block translation. Fourth, the relative importance of the A-rich and U-rich elements in controlling the level of translation differs. Fifth, none of our MOS 3'-UTR manipulations relieved translational repression before germinal vesicle breakdown. Sixth, all the MOS mRNA variants underwent polyadenylation during maturation. Whereas mutations to the hexanucleotide signal block both polyadenylation and translation, mutations to either the A-rich sequence or the U-rich elements block translation without fully blocking polyadenylation. We conclude that MOS mRNA translation in pig oocytes is subject to a more extensive series of controls than that in lower vertebrates.

Biodistribution and genotoxicity of transferrin-conjugated liposomes/DNA complexes in mice

Transferrin-conjugated liposomes (Tf-liposomes) were made and formulated with pCMVluc DNA to form a lipoplex. Among the various formulations studied, the Tf-liposome: pCMVluc DNA complex at a ratio of 5:1 (wt/wt) showed the highest transfection efficiency, which was twice that of LipofectinTM on HeLa cells. The maximum tolerated dose (MTD) of this lipoplex formulation from a single intravenous injection was over 10 mg/kg in healthy ICR mice. The RT-PCR results showed that the highest level of luciferase mRNA was detected in the lungs, followed by the liver, spleen, heart and kidneys, after an intravenous injection into mice. Two weeks after the injection, the levels of luciferase mRNA decreased gradually in the liver, spleen, heart, and kidney, but not in the lungs. The micro-array study showed that the cancer-related genes, including the bcl 6 gene, were highly up-regulated by the treatment with Tf-liposome/ pCMVluc DNA complex on HeLa cells, indicating that there were possible interactions between the host chromosomal DNA and the Tf-liposome within the cells. The results obtained from this study are expected to be useful for designing a safe and efficient gene delivery system using transferrin-conjugated liposomes.

Differences in the translation efficiency and mRNA stability mediated by 5′-UTR splice variants of human SP-A1 and SP-A2 genes

Surfactant protein A (SP-A) plays an important role in host defense, modulation of inflammatory processes, and surfactant-related functions of the lung. The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2. Several hSP-A 5'-untranslated region (UTR) splice variants for each gene have been characterized and shown to be translated in vitro and in vivo. In this report, we investigated the role of hSP-A 5'-UTR splice variants on SP-A production and molecular mechanisms involved. We used in vitro transient expression of hSP-A 5'-UTR constructs containing luciferase as the reporter gene and quantitative real-time PCR to study hSP-A 5'-UTR-mediated gene expression. We found that 1) the four (A'D', ABD, AB'D', and A'CD') 5'-UTR splice variants under study enhanced gene expression, by increasing luciferase activity from 2.5- to 19.5-fold and luciferase mRNA from 4.3- to 8.8-fold compared with the control vector that lacked hSP-A 5'-UTR; 2) all four 5'-UTR splice variants studied regulated mRNA stability. The ABD variant exhibited the lowest rate of mRNA decay compared with the other three constructs (A'D', AB'D', and A'CD'). These three constructs also exhibited significantly lower rate of mRNA decay compared with the control vector; 3) based on the indexes of translational efficiency (luciferase activity/mRNA), ABD and AB'D' exhibited higher translational efficiency compared with the control vector, whereas the translational efficiency of each A'D' and A'CD' was lower than that of the control vector. These findings indicate that the hSP-A 5'-UTR splice variants play an important role in both SP-A translation and mRNA stability.

The RNA-binding Protein IMP-3 Is a Translational Activator of Insulin-like Growth Factor II Leader-3 mRNA during Proliferation of Human K562 Leukemia Cells

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and beta-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or beta-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.

199. Efficient Inhibition of Bone Metastasis Via Systemic Delivery of siRNA Using Atelocollagen-Mediated Delivery System

Top of pageAbstract RNA interference has been one of the most powerful tool for genetic analysis, and much research on the development of siRNA for therapeutic use has progressed. Since siRNA shows a very low efficiency in gene silencing in vivo, various delivery methods have been investigated. However, there are no reports of efficient treatment of bone metastatic cancer by a systemic delivery of siRNA. We have demonstrated the excellent properties of an atelocollagen-mediated delivery system for nucleic acid medicines such as plasmids, antisense DNA and siRNA. Atelocollagen has been widely used because of its high biocompatibility and biodegradability. Last year, we reported that atelocollagen forms a complex with siRNA, and intratumor injection of the complex containing siRNA against FGF 4 mRNA inhibits growth of an orthotropically xenografted human nonseminomatous germ cell tumor. In this study, we demonstrated that the delivery system markedly enhanced the efficiency of siRNA in inhibiting bone metastasis through systemic administration. The siRNA/atelocollagen complex was formed by mixing siRNA with atelocollagen. The formation of the complex was confirmed by gel electrophoresis, and the complex can be administrated by conventional methods. To facilitate the detection of metastatic lesions and tumor regression as the effect of the caused by the complex, noninvasive optical imaging technologies and the bioluminescent human prostate carcinoma cell line PC-3M-luc-C6 were employed. An intracardiac injection of PC-3M-luc-C6 induced metastasis to the thorax, jaws and/or legs of mice. Prior to a metastasis inhibition study, we tested whether a systemic administration of the complex induces a gene silencing effect on metastatic sites. When the complex containing siRNA against luciferase mRNA was administered via the tail vein, bioluminescence was inhibited by 80|[ndash]|90% in the entire body including bone metastatic sites. In contrast, treatment with siRNA alone did not changed or slightly suppressed photon emission from tumor cells. The findings indicate that the complex delivered siRNA with high integrity and inhibited gene expression in metastatic sites including the bone. To inhibit bone metastasis, siRNAs against mRNAs related to cell growth were complexed with atelocollagen, and the complexes administered via the tail vein. When atelocollagen alone, siRNA alone or a non specific siRNA/atelocollagen complex was administered, a high metastasis was observed. On the other hand, bioluminescence intensity in mice administered the complexes of siRNA that inhibits cell growth was inhibited by 40|[ndash]|50% in the entire body including the bone. These findings indicate that the atelocollagen-mediated systemic delivery of siRNA could be a novel strategy for the inhibition of bone-metastatic prostate tumor growth. Furthermore, we confirmed that thecomplex did not induce an interferon response. In summary, an atelocollagen-mediated delivery system has great potential in significantly advancing the practical therapeutic applications of gene suppression using siRNAs.

584. Gene Delivery to Primary Neuronal Cells

Top of pageAbstract Neuronal protection under numerous clinical circumstances (i.e. trauma brain injury, stroke, ischemia and circulation arrested surgery, etc.) remains a tough challenge due to the paucity of effective clinical options. Transient gene delivery to CNS neuronal cells may offer an alternative approach to induce neuronal protection. We have previously shown widespread distribution, uptake, and expression in rat brain after non-viral, cationic lipid-mediated delivery of reporter gene vectors to the lateral ventricle 1,2. In order to further develop techniques for, and understand the mechanisms of, non-viral gene delivery, we conducted cationic lipid mediated gene delivery of fire fly luciferase mRNA and DNA to primary rat cortical cells to study the post-transfection transgene expression profiles. The delivery of mRNA resulted in a rapid onset of luciferase expression 1 hour after transfection, peaked at 5- 6 hours post transfection, and diminished to base line 12 hours after transfection. Compared to the delivery of mRNA, DNA delivery resulted in a much later onset of gene expression. Although the transgene expression after DNA delivery was generally one order of magnitude higher than after mRNA delivery, the earliest significant luciferase activity after DNA transfection did not appear until 7 hours after transfection. Peak expression following DNA delivery was 36-48 hours after transfection, and transgene expression persisted at a significant level for at least one week before it dropped to base line. Based on this observation, we attempted the delivery of mRNA and DNA vectors that encode for the heat shock protein gene HSP70. The inducible HSP70 is a chaperone protein and its expression has protective effects under stress conditions. HSP70 was delivered using non-viral cationic lipid-mediated techniques that we have developed to rat primary cortical cells, and analyzed by flow cytometry and immunohistochemistry. Consistent with the luciferase activity results, we detected evenly stained HSP70 expression 5 hours after the delivery of HSP70 mRNA, while expression after the delivery of HSP70 DNA appeared to peak at 36-48 hours.

Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells

The effect of RNA interference (RNAi) is generally more potent in Drosophila Schneider 2 (S2) cells than in mammalian cells. In mammalian cells, PolIII promoter-based DNA vectors can be used to express small interfering RNA (siRNA) or short hairpin RNA (shRNA); however, this has not been demonstrated in cultured Drosophila cells. Here we show that shRNAs transcribed from the Drosophila U6 promoter can efficiently trigger gene silencing in S2 cells. By targeting firefly luciferase mRNA, we assessed the efficacy of the shRNAs and examined the structural requirements for highly effective shRNAs. The silencing effect was dependent on the length of the stem region and the sequence of the loop region. Furthermore, we demonstrate that the expression of the endogenous cyclin E protein can be repressed by the U6 promoter-driven shRNAs. Drosophila U6 promoter-based shRNA expression systems may permit stable gene silencing in S2 cells.

Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteine in vivo and in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to "read-through" a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3'-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3'-untranslated region of the luciferase gene stimulated read-through by 6-11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p < 0.01) and decreased GPx1 enzyme activity by 45% (p = 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p < 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.