Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents.     

人大麻素受体启动子真核报告质粒的构建及活性分析

目的:利用双荧光素酶报告基因体系,根据大麻素受体1(cannabinoid receptor 1,cnr1)启动子序列不同部位的转录活性来初步确定其活性区域,为脊髓缺血耐受保护中cnr1高表达的转录调控的研究奠定基础。方法:从NCBI中获取cnr1基因转录起始点5’端向上约1800 bp的核苷酸序列,按照300 bp的距离间隔,设计6个不同长度的截短体PCR引物,以人全血基因组DNA为模板,分别扩增cnr1启动子区域的截短体片段,并克隆入荧光素酶报告基因pGL3-Basic质粒中。将含有不同截短体的重组质粒分别转染Hela、Jurket和A549细胞后行荧光素酶活性检测。根据不同截短体转录活性检测结果,确定cnr1启动子活性区域。结果:成功将cnr1启动子区6个不同长度(1800、1500、1200、900、600和300 bp)的截短体克隆入荧光素酶报告基因pGL3-Basic质粒。在3种细胞系Hela、Jurket和A549的荧光素酶活性检测均显示600 bp的截短体转录活性最强。结论:成功构建了cnr1启动子的报告基因重组质粒,初步证实-600 bp到-200 bp区为cnr1的启动子的活性区域,从而为进一步研究cnr1的转录调控奠定了基础。     

人B7H4启动子荧光素酶报告基因载体的构建及活性分析

为构建人B7H4基因启动子荧光素酶报告基因载体,以人外周血单个核细胞基因组DNA为模板,PCR扩增3条不同长度人B7H4启动子序列,并插入PGL3-Basic荧光素酶报告基因载体中;待测序验证后,将3个重组质粒及pRL-TK内参质粒分别共转染HEK-293T细胞,采用双荧光素酶报告基因系统检测其启动子活性。测序结果显示构建的3个人B7H4基因启动子重组载体序列正确;重组载体转染HEK-293T细胞,经双荧光素酶报告基因检测确定重组载体有启动子活性,其中PGL3-hB7H4-0.5kb重组载体的转录活性较高。本研究成功构建了3条含不同长度的B7H4启动子序列的荧光素酶报告基因系统,为后续分析人B7H4启动子的转录调控元件及肿瘤微环境中调控B7H4表达的作用因素等研究奠定了实验基础。     

Specific identification of Mycobacterium tuberculosis with the luciferase reporter mycobacteriophage: use of p-nitro-alpha-acetylamino-beta-hydroxy propiophenone.

We have previously described a luciferase reporter mycobacteriophage (LRP) assay that can detect Mycobacterium tuberculosis and characterize mycobacterial drug susceptibility patterns within 24 to 48 h in positive cultures. One drawback of this LRP protocol is the ability of the recombinant mycobacteriophage phAE40 to infect a variety of Mycobacterium species, thus limiting its specificity for the detection of M. tuberculosis. In this study, we have (i) explored the host range of phAE40, (ii) developed a modified LRP assay that exploits the selective inhibitory effect of the compound p-nitro-alpha-acetylamino-beta-hydroxy propiophenone (NAP) against members of the M. tuberculosis complex to differentiate between the tubercle bacillus and other mycobacterial species, and (iii) tested over 300 samples, including primary clinical isolates and drug-resistant strains of M. tuberculosis, demonstrating the ability of the NAP-modified LRP assay to identify M. tuberculosis complex organisms with high degrees of sensitivity and specificity.     

NOX4报告基因重组质粒的构建及表达调控初探

目的：构建NOx4荧光素酶报告基因质粒载体,探讨NOX4基因表达调控机制。方法：以细胞基因组DNA为模板作,采用PCR扩增NOX4基因上游调控序列（533bp）,插入质粒DGL3-basic载体,构建重组质粒pGL3-NOX4,重组质粒经酶切和测序鉴定。将pGL3-NOX4转染A549细胞,通过细胞因子刺激观察报告基因表达水平。结果：酶切及测序证实NOX4报告基因质粒构建成功。转染报告基因的A549细胞以细胞因子刺激,结果显示NOx4表达增强。结论：成功构建了NOX4荧光素酶报告基因质粒载体,为进一步研究NOX4基因的表达规律及生理功能奠定了基础。     

A novel luciferase and GFP dual reporter virus for rapid and convenient evaluation of hepatitis C virus replication

Herein, we describe the development of a monocistronic dual reporter virus for monitoring hepatitis C virus (HCV) replication. The recombinant construct encodes for the humanized Renilla luciferase (hRLuc) reporter gene inserted upstream of the viral open reading frame and a green fluorescent protein (GFP) gene inserted into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome. The viral RNA replicated efficiently in transfected cells and infectious virions could be produced without obvious attenuation of viral replication. The viral titer of the dual reporter virus was comparable to that of single reporter viruses. The expression levels of these two reporter genes correlated well with HCV replication in the presence or absence of antiviral agents. Moreover, because of the direct visibility of GFP fluorescence and the correlation between GFP positive cell numbers and hRLuc activity, the optimal time for measuring hRLuc activity was determined. This novel infectious system is a time saving and cost effective method for studying the interaction between viruses and host cells as well as for screening anti-HCV drugs.     

检测T-bet表达活性的荧光素酶报告基因质粒的构建和鉴定

目的 构建一个能应用于T-bet转录活性研究和大规模筛选的报告基因质粒.方法 以IFN-γ基因的CNS-22 T box结合位点序列为基础,人工合成顺式元件DNA(TbRE),重组连接到报告基因载体pLUC-MCS中.经测序验证后,转染293T细胞检测报告基因对T-bet的响应,并通过电泳迁移率检测(EMSA)证实T-bet与TbRE元件的特异结合.结果 成功构建了TbRE-LUC报告基因质粒.经荧光素酶活性检测证实外源表达的T-bet对报告基因的表达可以有20倍左右的激活,并且存在剂量效应,T-bet的表达量与报告基因的表达水平呈正相关.经电泳迁移率检测验证,T-bet可以与TbRE顺式反应元件特异性结合.结论 成功构建了T-bet转录活性相关的报告基因质粒TbRE-Luc,该报告基因系统反应灵敏,特异性强,可用于大规模筛选T-bet转录调控相关基因.     

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.     

Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis.

TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.     

The use of adenoviral vectors for genetic manipulation and analysis of primitive hematopoietic cells

Gene transfer into stem cells has long been studied as a means by which primitive hematopoietic cells could be characterized and manipulated. While a variety of strategies have been attempted, it still remains relatively difficult to perform direct stem cell analysis. In this review, we examine recent studies using adenovirus-based vectors as a means to achieve high-level gene transfer into primitive hematopoietic cell types.     

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.     

Separating fact from fiction: assessing the potential of modified adenovirus vectors for use in human gene therapy

One of the major hurdles to successful gene therapy of genetic and/or acquired disease is the ability to efficiently introduce a foreign gene into the tissue of interest and, in the case of some genetic diseases, achieve long-term expression of the transgene. Due to their ability to transduce a wide variety of cell types in a cell-cycle independent fashion, adenovirus (Ad)-based vectors have received considerable attention in recent years as delivery vehicles for multiple gene therapy applications. Effective use of early "first-generation" versions of these vectors was hampered by not only the induction of strong immune responses in the host to the Ad vector and transduced cells, but also to direct acute and chronic toxicity caused by the vector itself. Furthermore, transgene expression was typically transient, lasting only a few weeks. Despite these limitations, these vectors have been used in a number of human clinical trials, eliciting both interesting as well as controversial results, some of which are summarized herein. Because of these limitations, a number of advances in adenovirus "vectorology", manifested primarily as the development of multiply attenuated Ads and vectors deleted of all viral protein coding sequences, has resulted in vectors which retain all of the advantages of Ad vectors and, in addition, do not exhibit the deleterious characteristics associated with [E1-]deleted Ads. This review focuses on the current state of the art regarding the potential for human use of Ad-based vectors, and how the use of this vector continues to offer the potential for successful use as a gene delivery tool for the treatment of a great number of human genetic and non-genetic diseases.     

Cryopreservation of lymphocytes for use in in vitro assays of cellular immunity.

Human lymphocytes can be cryopreserved for the purpose of performing in vitro tests of cellular immunity. A systematic study of the conditions for freezing and recovering cells has shown that there is wide flexibility in cell concentrations of cryoprotective agent and the temperature and rate of dilution however, have definable optima. Cells frozen and thawed under optimal conditions retain their reactivity in MLC and can be used for sequential studies of immune responsiveness. Approximately 70% of viable and functional cells are recovered when the cells are frozen in 7.5--12.5% DMSO and no further cryoprotection is discernable when up to 50% serum is added to the freezing media. The temperature and rate of dilution are critical only in that cells diluted rapidly in THE COLD (10-fold in 2 min at 0 degrees C) are less responsive in MLC than are cells diluted slowly (10-fold in 10 min) in the cold or slowly or rapidly at room temperature.     

Trends in the use of medications with insomnia side effects and the implications for insomnia among US adults

Americans are increasingly consuming pharmaceuticals that although effective in treating their focal indication, include insomnia as a side effect. Regardless, no studies have documented trends in the concurrent use of these medications and their implications for insomnia among community‐dwelling adults. Using a nationally representative sample of US adults from the 1999–2016 National Health and Nutrition Examination Survey (NHANES), this study shows that the concurrent use of medications with insomnia as a potential side effect (“insomnia side effects” hereafter) has increased considerably in the past two decades. Between 1999 and 2016, the use of one and two or more medications with insomnia side effects increased by 66% and 164%, respectively. Compared to non‐users, respondents who took two or more of these medications were more likely to report insomnia symptoms (odds ratio [OR] = 1.78; 95% confidence interval [CI], 1.22 to 2.60), daytime sleepiness symptoms (OR = 1.73; 95% CI, 1.16 to 2.60) and difficulty with at least two daytime activities due to sleepiness or tiredness (OR = 1.96; 95% CI, 1.28 to 3.00). These findings highlight the need for insomnia screenings among patients who consume medications with insomnia side effects. They also emphasize the increased risks of insomnia associated with polypharmacy.     

Modulation of the immune response by immunoglobulin for intravenous use. II. Inhibitory effects of sera from treated patients

Sera were collected from patients with common varied immunodeficiency (CVI) prior to and following intravenous gamma-globulin (IVGG) infusion. Cultures of pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells from normal donors in medium containing post-IVGG infusion sera generated significantly fewer plaque-forming cells (PFC) than those cultures in medium containing the corresponding pre-IVGG infusion sera. However, preinfusion CVI sera were found to be similar to normal sera in their capacities to support PWM-induced PFC generation, despite the disparity in Ig levels between the two groups of sera. Furthermore, serum collected from a CVI patient 24 hr or more after IVGG infusion no longer possessed the same inhibitory capacity as serum collected 10 min after IVGG infusion despite elevated IgG levels compared to baseline. These studies suggest that IVGG infusion may induce an immunosuppressive effect which is transient in nature, raising the possibility of in vivo counterbalancing homeostatic mechanisms responding to this immune perturbation.     

Preliminary analysis of mortality associated with rituximab use in autoimmune diseases

Abstract

Normal antibodies and pathogenic autoantibodies are produced by B-cells and plasma cells. Rituximab is a chimeric monoclonal antibody that targets the CD20 molecule on cells that express them on their surface and kills them. Rituximab has been increasingly used to treat several autoimmune diseases. Studies on fatal outcomes associated with rituximab therapy are lacking. A comprehensive and detailed analysis in which the multiple factors that could contribute to a fatal outcome in all the autoimmune diseases in which rituximab has been used would be cumbersome, lack uniformity and would prove difficult in making certain definitive conclusions and comparisons, but more importantly it would not allow to provide specific precautions and recommendations to prevent mortality. Hence, autoimmune mucocutaneous blistering diseases (AMBD) were used as model to study fatal outcomes in patients treated with rituximab between 2000 and 2013, using uniform 13 criteria. Fatal outcomes were found in 14 patients with autoimmune blistering diseases out of 134 patients (10.4%). Patients died due to infections (75%), gastrointestinal (17%) and cardiac events (8%). Causes of death were reported in 101 patients with other autoimmune diseases out of 4320 with a mortality rate of 2.4%. Among them, 44 patients (43.6%) died from infections. A statistical analysis of the data demonstrated that a statistically significant higher mortality rate was observed in patients with AMBD compared to patients with other autoimmune diseases. Similarly, a statistically significant higher rate of death due to infections was reported in patients with AMBD compared to patients with other autoimmune diseases. Use of systemic corticosteroids and immunosuppressive agents as concomitant therapy with rituximab enhanced immunosuppression. In many patients, B-cells were depleted for prolonged periods, even after clinical recovery was observed. Although its main action is depletion of B-cells, rituximab has a significant impact on the immune and inflammatory systems, directly and indirectly and thus enhances susceptibility to infection. These preliminary data suggests that physicians using rituximab to treat autoimmune diseases should monitor their patients closely, especially their B-cell levels until they return to normal, be vigilant for possible sources of infection, and be aware of potential fatal outcomes.     

Gateway compatible vectors for analysis of gene function in the zebrafish.

The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos.     

Hepatitis C virus infection in post-transfusion hepatitis. An analysis with first- and second-generation assays.

BACKGROUND. The causes of post-transfusion non-A, non-B hepatitis are still not fully defined, nor is it clear how accurate the tests are that are used to screen blood donors for hepatitis C virus (HCV) and to diagnose post-transfusion hepatitis caused by infected blood. METHODS. We used two first-generation enzyme-linked immunoassays (EIAs) and one second-generation immunoassay to test for anti-HCV antibodies in serum samples collected between 1976 and 1979 in the Transfusion-Transmitted Viruses Study (from 1247 patients who underwent transfusion and 1235 matched control subjects who did not receive transfusions). We tested serum collected before and after infection from the patients in whom non-A, non-B hepatitis developed, serum from their blood donors, and serum from 41 of the control subjects who had hepatitis unrelated to transfusion. RESULTS. Of the 115 patients in whom post-transfusion non-A, non-B hepatitis developed, the initial serum samples of 111 were anti-HCV-negative; after hepatitis developed in these 111 patients, the first-generation EIAs detected anti-HCV in 51 (46 percent), and the second-generation assay detected anti-HCV in an additional 16 (14 percent), for a total of 60 percent. Of 40 controls, 37 were anti-HCV-negative initially, and none seroconverted after hepatitis developed. If the 3 percent rate of non-A, non-B, non-C hepatitis among the controls (37 of 1235) was applied to the 1247 transfusion recipients, only 74 of the 111 cases of hepatitis were attributable to the transfusion. Thus, 91 percent (67 of 74) of the cases of post-transfusion hepatitis were caused by HCV. Of the 99 donors, 60 were HCV-positive (9 on second-generation tests only) and 39 were not. CONCLUSIONS. Nearly all cases of non-A, non-B post-transfusion hepatitis are caused by HCV. Screening with a second-generation assay improves the rate of detection of HCV infection in patients with post-transfusion hepatitis and in blood donors. The use of this test showed a 3.6 percent risk of non-A, non-B, non-C hepatitis, which was not significantly different from the rate in the controls (3.0 percent).