Antisense activity detection by inhibition of fluorescence resonance energy transfer.

Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.     

Rapid turnover of the "functional" Na(+)-Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach

Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na(+)-Ca2+ exchanger (NCX1) to correlate suppression of the Na(+)-Ca2+ exchange function with down-regulation of NCX1 protein expression. In a de-novo expression system (Sf9 cells), a decrease in both, NCX1 mRNA and protein after AS-ODN application was paralleled by diminished NCX1 activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in whole-cell extracts of these cells treated with AS-ODNs, the amount of NCX1 protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCX1 function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCX1 protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins.     

Antisense downregulation of SARS-CoV gene expression in Vero E6 cells

BACKGROUND: Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). It is an enveloped, single-stranded, plus-sense RNA virus with a genome of approximately 30 kb. The structural proteins E, M and N of SARS-CoV play important roles during host cell entry and viral morphogenesis and release. Therefore, we have studied whether expression of these structural proteins can be down-regulated using an antisense technique. METHODS: Vero E6 cells were transfected with plasmid constructs containing exons of the SARS-CoV structural protein E, M or N genes or their exons in frame with the reporter protein EGFP. The transfected cell cultures were treated with antisense phosphorothioated oligonucleotides (antisense PS-ODN, 20mer) or a control oligonucleotide by addition to the culture medium. RESULTS: Among a total of 26 antisense PS-ODNs targeting E, M and N genes, we obtained six antisense PS-ODNs which could sequence-specifically reduce target genes expression by over 90% at the concentration of 50 microM in the cell culture medium tested by RT-PCR. The antisense effect was further proved by down-regulating the expression of the fusion proteins containing the structural proteins E, M or N in frame with the reporter protein EGFP. In Vero E6 cells, the antisense effect was dependent on the concentrations of the antisense PS-ODNs in a range of 0-10 microM or 0-30 microM. CONCLUSIONS: The antisense PS-ODNs are effective in downregulation of SARS. The findings indicate that antisense knockdown of SARS could be a useful strategy for treatment of SARS, and could also be suitable for studies of the pathological function of SARS genes in a cellular model system.     

Trafficking of intracerebroventricularly injected antisense oligonucleotides in the mouse brain

Intracerebroventricular (icv) delivery of therapeutic molecules directly into the brain parenchyma has attracted considerable attention because of the advantage of bypassing the blood-brain barrier. Exogenous icv administration of antisense oligodeoxynucleotides (AS-ODNs) has been implicated in modifying gene expression within the targeted brain area. The biodistribution, tissue penetration, and stability of exogenously administered AS-ODNs are the major determinants with regard to their potential utility as agents for modifying gene expression. This report examined the distribution and clearance of labeled AS-ODNs with the aim of exploring the feasibility of icv administration of AS-ODNs as a targeted treatment approach to Alzheimer's disease. A single icv injection of fluorescein-labeled 2'-O-(methoxy) ethyl (2'MOE) ribosyl-modified AS-ODNs directed at the beta-secretase cleavage site of beta-amyloid precursor protein (APP) mRNA into the mouse brain showed rapid uptake by 15 minutes, overall gradual spread and retention by 30 minutes to 3 hours, and complete clearance by 8 hours postinjection. Labeled AS-ODNs were observed to penetrate across the cell membrane and accumulate in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cell populations. Current study provides a basic pattern of uptake, distribution, and stability of AS-ODNs in the mouse brain.     

两株绿脓杆菌对石油污染土壤的修复作用

本文旨在研究环境条件下微生物对石油污染土壤的修复情况。从矿井周边土样定向筛选出两株绿脓杆菌,摇瓶降解实验发现,两菌混合培养10 d原油降解率达到95.67%,比单菌培养提高至少32%,即两菌对原油降解具有协同作用。根据降解实验结果制备了混合修复菌剂,并且人工构建石油污染场地,展开中试场地修复试验,模拟不同的操作条件下土壤中原油的降解情况。经60 d修复发现,添加了菌剂的场地,石油烃含量下降趋势明显,每克土壤中石油烃含量从初始的0.8%降至0.1%–0.3%,其中额外添加有机肥作为补充碳氮源的场地,总石油烃降解率最高,达到85.28%。而未添加菌剂的对照组石油烃含量仅减少25.85%。     

Antisense oligodeoxynucleotides targeting internal exon sequences efficiently regulate TNF-alpha expression.

ABSTRACT Exon sequences upstream of splice sites play a critical role in mRNA processing, which is dependent on spliceosome interactions with these sites. Using antisense oligodeoxynucleotides (ODN), we targeted these and other sequences of the proinflammatory tumor necrosis factor-alpha (TNF-alpha) gene because it is multiply spliced and has been difficult to regulate with ODN in the past. ODN targeting exon sequences upstream of the donor splice sites of internal exons 2 (ORF4) and 3 (ORF6) significantly reduced TNF-alpha levels in stimulated U937 cells by 62%+/-7% and 51%+/-9%, respectively, in a dose-dependent manner but did not affect interleukin-6 (IL-6) levels. In contrast, ODN targeting the exon sequences downstream of the acceptor splice sites of exons 1, 2, and 3 failed to reduce TNF-alpha levels significantly under the same conditions. End-phosphorothioated ORF4 (ORF4-PE) significantly reduced TNF-alpha mRNA levels by greater than 80% (p < 0.001) and protein levels by 60% (p < 0.001) in U937 cells. ORF4-PE reduced newly synthesized TNF-alpha protein levels by >80% in lipopolysaccharide (LPS)-stimulated human macrophages, by greater than 60% in phorbol myristate acetate/phyto-hemagglutinin (PMA/PHA)-stimulated human peripheral blood mononuclear cells (PBMC), and by approximately 50% in LPS-stimulated murine monocytes. These results suggest that exon sequences flanking donor splice sites are highly susceptible target domains for antisense inhibition of TNF-alpha gene expression.     

玉米大斑病菌STK1与EGFP融合基因载体的构建及其在毕赤酵母中的表达

STK1基因是玉米大斑病菌调控分生孢子发育、渗透胁迫调节和致病性的重要MAPK基因。本文首先构建了含有增强型绿色荧光蛋白基因(EGFP)的毕赤酵母GSS115(Pichia pastoris GS115)表达载体p PIC3.5K-EGFP,再以玉米大班病菌模式菌株01-23的菌丝c DNA为模板,PCR扩增STK1基因,克隆到p PIC3.5K-EGFP,构建了STK1-EGFP融合基因的GS115表达载体p PIC3.5K-STK1-EGFP。利用电击转化法将该融合基因表达载体转化到GS115感受态细胞内,利用MD培养基筛选、PCR鉴定,获得了STK1-EGFP融合基因的毕赤酵母转化子。通过RT-PCR和荧光观察,发现STK1基因和EGFP基因均可以高效稳定地表达。另外,在试验中我们还发现,在STK1基因起始密码子前加入Kozak序列可以使STK1-EGFP融合基因的表达强度增强4.8倍。以上研究结果为STK1基因表达蛋白的亚细胞功能定位和抗体制备奠定了基础。     

Accelerated assessing of antisense RNA efficacy using a chimeric enhanced green fluorescent protein-antisense RNA-producing vector

The selection of suitable parts of a gene as antisense RNA sequences is largely a matter of trial and error and, as a consequence, a rather time-consuming process. In this study, we present a rapid and reproducible method to bypass this protracted procedure by using a chimeric enhanced green fluorescent protein (EGFP)-antisense RNA-producing vector. The combination of a reporter gene and antisense RNA allows easy measurement by flow cytometry of antisense RNA efficacy in successfully transfected cells shortly after transfection. Four chimeric EGFP-p185c-erbB-2-antisense RNA vectors were constructed and transfected into the p185-c-erbB-2-overexpressing cell line SKBR3. Within 1 week, we were able to estimate the inhibitory capacities of the different antisense RNA sequences used in this study. Our results strongly suggest that a chimeric EGFP-antisense RNA vector is an appropriate tool to expedite the laboratory work and time in screening the efficacy of antisense RNA strategies.     

In vitro transfer of antisense oligodeoxynucleotides into coronary endothelial cells by ultrasound

Since antisense oligodeoxynucleotides (AS-ODNs) have been recognized as a new generation of putative therapeutic agents, we established a delivery technique that could transfect AS-ODNs, which are designed for endothelin type B receptor (ETB), into cultured human coronary endothelial cells (HCECs) by exposure to ultrasound in the presence of echo contrast microbubbles. Ultrasound offers several advantages such as being nontoxic, nonantigenic and providing rapid gene transfer. We standardized the optimal conditions, which consisted of 2 x 10(6) cells suspended in phosphate buffer with 900nM ODN, 50 microl of echo contrast microbubbles (Optison), and ultrasound exposure (1.0 W/cm(2), 10% duty cycle, and 10s duration). The percentage of transfected cells was 25.2+/-2.0% after ultrasound treatment. This is the first demonstration of the use of the ultrasound exposure technique in conjunction with microbubbles in HCECs.     

Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.     

Inhibition of CD4 expression by antisense oligonucleotides in PMA-treated lymphocytes

To decrease CD4 expression on T helper (Th) lymphocyte surface, antisense oligonucleotides (AS-ODNs), delivered by the cationic liposome DOTAP, were assayed in vitro on rat spleen lymphocytes. Four 21-mer ODNs (AS-CD4-1, AS-CD4-2, AS-CD4-3, and AS-CD4-4) directed against the translation start region of the cd4 gene were designed. AS-CD4-1 was phosphorothioate (PS)-modified in each base, and the other three were PS-modified at both ends and in the internal pyrimidine residues. Four ODN controls (fully PS-modified ODN-A and partially modified ODN-B, ODN-C, and ODN-D) were also assayed. CD4 resynthesis was stimulated by treatment with phorbol 12-myristate 13-acetate (PMA) at the same time as the incubations with the ODN. After 24 hours of treatment, CD4 expression was measured by immunofluorescence staining and flow cytometry. CD4 reexpression in rat PMA-treated lymphocytes was counteracted by 40% by means of AS-CD4-2 and AS-CD4-4 treatments. On the other hand, AS-CD4-3 produced only 20% inhibition, similar to that produced by ODN-B, and AS-CD4-1 did not have any significant effect compared with control ODNs. Both AS-CD4-2 and AS-CD4-4 decreased CD4 mRNA, as determined by RT-PCR, and in addition, they did not affect the expression of other surface lymphocyte molecules. Inhibition of surface CD4 expression remained at least 72 hours. The addition of both AS-ODNs did not further increase the effect obtained separately by each AS-ODN. Treatment of rat PMA-lymphocytes with two concentrations of AS-CD4-2 and AS-CD4-4 added 24 hours apart did not further decrease CD4 expression. In summary, AS-CD4-2 and AS-CD4-4 could constitute a good strategy to inhibit CD4 expression on Th lymphocytes and modulate their function.     

Development of an assay for beta-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening.     

Expression and characterization of a recombinant Cry1Ac crystal protein with enhanced green fluorescent protein in acrystalliferous Bacillus thuringiensis

AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined. METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae. CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells.     

Production of cell lines secreting TAT fusion proteins.

Transduction of proteins and other macromolecules constitutes a potent technology to analyze cell functions and to achieve therapeutic interventions. In general, fusion proteins with protein transduction domains, such as TAT, are produced in a bacterial expression system. Here we describe the generation of a mammalian expression vector coding for TAT-EGFP fusion protein. Transfection of CHO-K1 cells by this vector and subsequent selection by Zeocin resulted in cell lines that express and secrete EGFP, a variant of the green fluorescent protein GFP. The ultimate cell line was produced by first cloning the stable integrants and subsequent selection of EGFP-expressing cells by flow cytometric sorting. In the resulting cell line approximately 98% of cells express EGFP. Using the same methodology, we generated cell lines that express DsRed fluorescent protein. The advantages of using such a mammalian expression system include the ease of generating TAT fusion proteins and the potential for sustained production of such proteins in vitro and, potentially, in vivo.