A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency

CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.     

An in vivo dual-reporter system of cyanobacteria using two railroad-worm luciferases with different color emissions

In vivo genetic reporter systems using luciferase enzymes enable the real-time monitoring of gene expression in living cells. We have challenged concurrent monitoring of two independent promoter activities within the same cells to precisely compare their characteristics in vivo. In this report, we describe a simple dual-reporter system capable of simultaneously monitoring two promoter activities in living cyanobacterial cells. Two railroad-worm luciferases catalyzing the bioluminescent emissions of different colors served as the dual reporters; each emission was successfully separated by interference filters to estimate the individual bioluminescence signals using photomultiplier tubes. Using this system, we clearly demonstrated the difference in the expression profiles between promoters in the same cells.     

Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-α) gene

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-alpha is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-alpha in macrophages, we attempt to establish a reporter system to assess TNF-alpha gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-alpha promoter and 3'-untranslated region (3'-UTR). The TNF-alpha-luciferase reporter cell line is used for assessing the expression of TNF-alpha gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-alpha such as dexamethasone and emodin, and also for measuring change of expression of TNF-alpha gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-alpha. The luciferase activity correlated well with the ELISA results for TNF-alpha production, therefore, the TNF-alpha-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-alpha gene.     

Bioluminescent detection of endotoxin effects on HIV-1 LTR-driven transcription in vivo.

We investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor kappaB (NF-kappaB)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5-6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3-6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.     

Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.     

Single nucleotide polymorphism analysis and functional characterization of the human Ah receptor (AhR) gene promoter

The aryl hydrocarbon receptor (AhR) mediates biological and toxicological actions of e.g., halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Although much is known about the biochemical and molecular mechanisms of AhR action, little is known about the control of the expression of the AhR gene itself. Therefore, we aimed at the identification and characterization of regions important for constitutive AhR gene expression. First, we screened 2.6 kb of the 5(')-flanking region of the AhR gene in 91 healthy Caucasian volunteers for naturally occurring genetic variants. Seven variants were detected. However, they do not seem to influence AhR gene expression in lymphocytes. Using a 2.7 kb AhR promoter luciferase reporter gene construct and various deletion constructs, a putative regulatory region was identified and characterized further by electrophoretic mobility shift assays and site-directed mutagenesis. These investigations were confirmed by cotransfection experiments in Drosophila SL2 cells. The obtained results prove an involvement of Sp1 in AhR gene regulation.     

Method for screening ecdysone-inducible stable cell lines

Ecdysone-inducible systems are useful tools to study the function of various genes in different types of mammalian cells. However, it is technically difficult to establish stable cell lines. This is partly because the conditional expression system requires the expression of two or more components driven by different kinds of promoters. In this paper, we describe the use of a luciferase reporter gene system for rapid screening of cell lines that express the ecdysone and retinoid X receptors. Using this system, two human stable colon cancer cell lines, SW480/VgRXR and HCT116/VgRXR, were successfully generated. The expression of these receptors remained high after six months of continuous culturing. A tight regulation of gene induction in SW480/VgRXR was observed using 2 microM Ponasterone A. The gene induction was rapid and persistent. Our results demonstrated the advantage of establishing cell lines that continuously express high levels of ecdysone receptor proteins.     

呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析

目的：探讨呼吸道黏蛋白（mucin,MUC）5AC基因5'上游序列顺式调控元件在中性粒细胞弹力酶(neutrophil elastase , NE)诱导MUC5AC基因转录表达的调控机制。方法：应用DNA重组技术,构建含萤光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒。采用定点突变技术,在嵌合质粒的基础上构建MUC5AC启动子区特殊蛋白（specificity protein）-1和核因子(nuclear factor, NF)-κB结合位点单独突变体,并测定NE刺激的转染细胞荧光素酶相对活性。结果：成功构建了4种含有不同长度MUC5AC基因启动子序列的荧光索酶报告基因质粒。含有启动子序列-1330bp、-689bp、-324bp的嵌合质粒荧光素酶相对光强度较对照组均显著增加,而含有启动子序列-64bp的嵌合质粒荧光素酶相对光强度与对照组相比差异无统计学意义。NE可诱导含有MUC5AC启动子区NF-кB结合位点单独突变体（pGL3E-MUC5AC-NF-кB-MU）荧光素酶相对光强度增加,而NE不能诱导Sp-l结合位点单独突变体（pGL3E-MUC5AC-SP-1-MU）荧光素酶表达增加。结论：MUC5AC 5'上游序列中-324～-64位点存在参与NE诱导MUC5AC基因表达的重要调控元件,位于此区域的顺式作用元件Sp-1位点在NE诱导MUC5AC基因表达机制中起重要作用,该位点可能作为靶向性基因治疗的关键调控元件。     

Reporter gene vectors and assays

Gene reporter systems play a key role in gene expression and regulation studies. This review describes the ideal reporter systems, including reporter expression vector design. It summarizes the many uses of genetic reporters and outlines the currently available and commonly used reporter systems. Each system is described in terms of the reporter gene, the protein it encodes, and the assays available for detecting presence of the reporter. In addition, each reporter system is analyzed in terms of its recommended uses, advantages, and limitations.     

法尼醇X受体激动剂细胞筛选体系的建立与优化

本研究旨在建立一种基于双荧光素酶报告基因检测体系的法尼醇X受体(farnesoid X receptor, FXR)激动剂细胞筛选体系,以满足对FXR激动剂先导化合物的高通量筛选。通过在报告基因质粒pGL4-luc2P-Hygro中的萤火虫荧光素酶(firefly luciferase,Luc)基因上游克隆并插入来自FXR靶基因的FXR反应元件(FXR response element,FXRE)片段,构建用于筛选FXR激动剂的报告基因质粒,并结合海肾荧光素酶内参质粒,建立能够有效反映药物对FXR激动效应的双荧光素酶报告基因细胞检测体系。通过一系列优化实验,比较了过表达RXR、鼠源和人源FXR、不同的FXRE片段、FXR过表达质粒与报告基因质粒的混合比对筛选体系诱导效率和灵敏度的影响。根据上述结果,最终确定了优化条件,优化后体系Z因子达到0.83。本研究建立了一种用于FXR激动剂筛选的改良的基于双荧光素酶报告基因检测体系的细胞筛选体系,其主要特征在于,使用多段FXR靶基因上的FXRE片段叠加组成一种新型的增强型FXRE元件,而非传统的反向重复序列-1 (inverted repeats...     

Cloning and mutagenetic modification of the firefly luciferase gene and its use for bioluminescence microscopy of engrailed expression during Drosophila metamorphosis

Bioluminescence microscopy is an area attracting considerable interest in the field of cell biology because it offers several advantages over fluorescence microscopy, including no requirement for excitation light and being phototoxicity free. This method requires brighter luciferase for imaging; however, suitable genetic resource material for this purpose is not available at present. To achieve brighter bioluminescence microscopy, we developed a new firefly luciferase. Using the brighter luciferase, a reporter strain of Drosophila Gal4-UAS (Upstream Activating Sequence) system was constructed. This system demonstrated the expression pattern of engrailed, which is a segment polarity gene, during Drosophila metamorphosis by bioluminescence microscopy, and revealed drastic spatiotemporal change in the engrailed expression pattern during head eversion in the early stage of pupation.