Reporter gene systems for the identification and characterization of cancer stem cells

Cancer stem cells(CSCs)are tumor cells that share functional characteristics with normal and embryonic stem cells.CSCs have increased tumor-initiating capacity and metastatic potential and lower sensitivity to chemo-and radiotherapy,with important roles in tumor progression and the response to therapy.Thus,a current goal of cancer research is to eliminate CSCs,necessitating an adequate phenotypic and functional characterization of CSCs.Strategies have been developed to identify,enrich,and track CSCs,many of which distinguish CSCs by evaluating the expression of surface markers,the initiation of specific signaling pathways,and the activation of master transcription factors that control stemness in normal cells.We review and discuss the use of reporter gene systems for identifying CSCs.Reporters that are under the control of aldehyde dehydrogenase 1A1,CD133,Notch,Nanog homeobox,Sex-determining region Y-box 2,and POU class 5 homeobox can be used to identify CSCs in many tumor types,track cells in real time,and screen for drugs.Thus,reporter gene systems,in combination with in vitro and in vivo functional assays,can assess changes in the CSCs pool.We present relevant examples of these systems in the evaluation of experimental CSCs-targeting therapeutics,demonstrating their value in CSCs research.

Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene’s expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.

Red fluorescent protein (DsRed2), an ideal reporter for cotton genetic transformation and molecular breeding

Genes encoding reporter proteins are used as visual marker-assisted tools in genetic transformation as well as plant breeding. In this study, the red fluorescent protein identified in Discosoma sp. coral(DsRed2) was successfully used as a visual marker for cotton genetic engineering. DsRed2 was successfully expressed in two cotton cultivars,JIN668 and YZ1, driven by the Ca MV-35 S promoter via the Agrobacterium-mediated transformation. Our results suggest that DsRed2 expression provides an early-stage selection tool for the transgenic calli via visual observation. Red fluorescence can be detected not only in callus and somatic embryos but also in most tissues and organs of mature plants. The transgenic line Yz-2-DsRed2 was crossed with four different cotton cultivars to assess the transgene heritability and stability in different genetic backgrounds.The heritability of the red color was highly stable when Yz-2-DsRed2 was used as a male parent. The DsRed2 gene expressed 100% in the F_1 hybrids. To investigate the relationship between DsRed2 transcription and DNA methylation, a methylation-specific PCR approach was applied to the Ca MV-35 S promoter region. The results showed a negative association between DNA methylation level in the promoter region and the transgene transcription.Taken together, these findings suggest DsRed2 a visual reporter gene for cotton genetic transformation and molecular breeding programs.

Construction and Identification of the Adenoviral Vector with Dual Reporter Gene for Multimodality Molecular Imaging

Summary： In this study, the recombinant adenovirus （Ad） vector containing dual reporter gene [i.e. human transferrin receptor gene （TFRC） and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance （MR） and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction （PCR） and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector （pAdeno） were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colo- rectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor （TfR） and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirns was constructed successfully, and the virus titer was 1.6x10^10 pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly （P〈0.01）. It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for in vivo tracing target cells in multimodality imaging.

Tetracysteine as a Reporter for Gene Therapy

Objective To study the feasibility of using tetracysteine （TC） reporter in gene therapy. Methods Effects of TC reporter and conventional reporter genes encoding green fluorescence protein （GFP） and luciferase （Luc） on expression and function of the therapeutic gene MGMT^P140K were compared. Cytotoxicity and drug resistance were studied by Western blot. TC reporter used in therapy was analyzed by flow cytometry （FCM）. Results The TC reporter had no toxicity to cells and neither affected the expression or activity of therapeutic gene as compared to GFP and Luc. TC could be used in blood sample detection. Conclusion TC is a new kind of reporter gene for lentiviral vector in future gene therapy.

Overview of the reporter genes and reporter mouse models

Reporter genes are widely applied in biotechnology and biomedical research owning to their easy observation and lack of toxicity. Taking advantage of the reporter genes in conjunction with imaging technologies, a large number of reporter mouse models have been generated. Reporter mouse models provide systems that enable the studies of live cell imaging, cell lineage tracing, immunological research and cancers etc. in vivo. In this review, we describe the types of different reporter genes and reporter mouse models including, random reporter strains, Cre reporter strains and ROSA26 reporter strains. Collectively, these reporter mouse models have broadened scientific inquires and provided potential strategies for generation of novel reporter animal models with enhanced capabilities.

VISUALIZATION OF HEAD AND NECK CANCER MODELS WITH A TRIPLE FUSION REPORTER GENE

The development of experimental animal models for head and neck tumors generally rely on the biol uminescence imaging to achieve the dynamic monitoring of the tumor growth and metastasis due to the complicated anatomical structures.Since the bioluminescence imaging is largely affected by the intracellular luciferase expression level and external D-luciferin concentrations,its imaging accuracy requires further confirmation.Here,a new triple fusion reportelr gene,which consists of a herpes simplex virus type 1 thymidine kinase(TK)gene for radioactive imaging,a far-red fuorescent protein(mLumin)gene for fuorescent imaging,and a firefly luciferase gene for bioluminescence imaging,was introduced for in vrivo observation of the head and neck tumors through multi-modality imaging.Results show that fuorescence and bioluminescence signals from mLumin and luciferase,respectively,were clearly observed in tumor cells,and TK could activate suicide pathway of the cells in the presence of nucleotide analog-ganciclovir(GCV),demonstrating the effecti veness of individual functions of each gene.Moreover,subcutaneous and metastasis animal models for head and neck tumors using the fusion reporter gene-expressing cell lines were established,allowing multi-modality imaging in vio.Together,the established tumor models of head and neck cancer based on the newly developed triple fusion reporter gene are ideal for monitoring tumor growth,assessing the drug therapeutic efficacy and verifying the effec-tiveness of new treatments.

3' Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells （DCMECs）. The miR-139 targeting GHR 3＇ UTR was predicted by Target Scan 5.1 software, 3＇ UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3＇ UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3＇ UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.

SPECT imaging of cardiac reporter gene expression in living rabbits

This work is to demonstrate feasibility of imaging the expression of herpes simplex virus 1-thymidine kinase (HSV1-tk) reporter gene in rabbits myocardium by using the reporter probe 131I-2'-fluoro-2'-deoxy-1-β-D- arabinofuranosyl-5-iodouracil (131I-FIAU) and SPECT. Rabbits of the study group received intramyocardial injection of Ad5-tk and control group received aseptic saline injection. Two sets of experiments were performed on the study group. Rabbits of the 1st set were injected with 131I-FIAU 600 μCi at Day 2 after intramyocardial transfection of Ad5-tk in 1×109, 5×108, 1×108, 5×107 and 1×107 pfu, and heart SPECT imaging was done at different hours. Rabbits of the 2nd were transferred various titers of Ad5-tk (1×109, 5×108, 1×108, 5×107, 1×107 pfu) to determine the threshold and optimal viral titer needed for detection of gene expression. Two days later, 131I-FIAU was injected and heart SPECT imaging was performed at 6, 24 and 48 h, before killing them for gamma counting of the hearts. Reverse tran- scription-polymerase chain reaction (RT-PCR) was used to verify the transferred HSV1-tk gene expression. Semi-quantitative analysis derived of region of interest (ROI) of SPECT images and RT-PCR images was performed and the relationship of SPECT images with ex vivo gamma counting and mRNA level were evaluated. SPECT images conformed 131I-FIAU accumulation in rabbits injected with Ad5-tk in the anterolateral wall. The optimal images qual- ity was obtained at 24~48 h for different viral titers. The highest radioactivity in the focal myocardium was seen at 6 h, and then declined with time. The threshold was 5×107 pfu of virus titer. The result could be set better in 1~5×108 pfu by SPECT analysis and gamma counting. ROI-derived semi-quantitative study on SPECT images correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The HSV1-tk/131I-FIAU reporter gene/reporter probe system is feasible for cardiac SPECT reporter gene imaging. The optimal Ad5-tk titer is 1~5×108 pfu and optimal imaging time is 24~48 h after transferred Ad5-tk in rabbit. The imaging of transgene expression in heart might be used for noninvasive imaging of gene therapy in cardiac diseases in human.

Nonsense variant of ATP8B1 gene in heterozygosis and benign recurrent intrahepatic cholestasis: A case report and review of literature

BACKGROUND Benign recurrent intrahepatic cholestasis is a genetic disorder with recurrent cholestatic jaundice due to ATP8B1 and ABCB11 gene mutations encoding for hepato-canalicular transporters.Herein,we firstly provide the evidence that a nonsense variant of ATP8B1 gene(c.1558A>T)in heterozygous form is involved in BRIC pathogenesis.CASE SUMMARY A 29-year-old male showed severe jaundice and laboratory tests consistent with intrahepatic cholestasis despite normal gamma-glutamyltranspeptidase.Acute and chronic liver diseases with viral,metabolic and autoimmune etiology were excluded.Normal intra/extra-hepatic bile ducts were demonstrated by magnetic resonance.Liver biopsy showed:Cholestasis in the centrilobular and intermediate zones with bile plugs and intra-hepatocyte pigment,Kupffer’s cell activation/hyperplasia and preserved biliary ducts.Being satisfied benign recurrent intrahepatic cholestasis diagnostic criteria,ATP8B1 and ABCB11 gene analysis was performed.Surprisingly,we found a novel nonsense variant of ATP8B1 gene(c.1558A>T)in heterozygosis.The variant was confirmed by Sanger sequencing following a standard protocol and tested for familial segregation,showing a maternal inheritance.Immunohistochemistry confirmed a significant reduction of mutated gene related protein(familial intrahepatic cholestasis 1).The patient was treated with ursodeoxycholic acid 15 mg/kg per day and colestyramine 8 g daily with total bilirubin decrease and normalization at the 6th and 12th mo.CONCLUSION A genetic abnormality,different from those already known,could be involved in familial intrahepatic cholestatic disorders and/or pro-cholestatic genetic predisposition,thus encouraging further mutation detection in this field.

Broad Hormonal Responses Induced by Aluminum in Roots of Dwarf Transgenics of Solanum lycopersicum L. cv “Micro-Tom”

The spatial pattern distribution of plant hormones in response to aluminum (Al) toxicity in roots remains to be shown. This study was performed to assess the root hormonal accumulation and gene expression in response to Al toxicity in five transgenic miniature dwarf tomatoes cv. Micro-Tom (MT). MT and MT transgenics to acid indole acetic, cytokinin, gibberellin, abscisic acid and ethylene were cultivated in nutrient solutions containing different Al concentrations. Root growth elongation was measured and cellular damage was visualized by staining Evans’s blue. The GUS reporter gene staining technique was used to visualize hormonal changes in MT apex root tissues. Data indicated that the MT is sensitive to Al that induced significant growth inhibition and cellular damage. Al concentration of 27 μM was significantly toxic, inducing root apex darkening and inhibition of root development. The qualitative evaluation of GUS reporter gene expression showed intense crosstalk among all hormones studied, underscoring the complexity of signaling induced by Al in apex roots. Results point out to a major understanding of the hormonal signaling in response to Al toxicity, which may induce a change of root growth and architecture with growth inhibition and cell constraints modulated by all different hormones evaluated.

孤立性心房颤动致病基因TBX20新突变的发现及功能研究

目的:寻找孤立性心房颤动(房颤)致病基因TBX20新突变并研究其功能。方法:测序分析182例孤立性房颤患者和236名无房颤对照者的TBX20基因,以发现新的致房颤突变。克隆人TBX20基因,构建野生型TBX20表达质粒,通过定点诱变制备突变型TBX20表达质粒,借助脂质体将表达质粒转染HeLa细胞,应用双荧光素酶报告基因分析系统研究突变体的功能特性。结果:在1例散发性孤立性房颤患者中发现TBX20基因新突变,即NM_001077653.2:c.706A>T;p(.Lys236*)突变。该突变不存在于其他孤立性房颤患者和对照者。功能研究显示突变型TBX20对靶基因KCNH2的转录激活作用丧失。结论:TBX20基因功能障碍可能是部分房颤患者的分子病因,这对房颤的精准防治有潜在临床意义。

强直性肌营养不良1型的临床特征和遗传学特点(附1家系报告)

目的探讨强直性肌营养不良1型(DM1)的临床特征和遗传学特点。方法对苏州大学附属第一医院2023年5月收治的DM1患者及其家系成员进行体格检查、EMG、肌肉活检和基因检测等检查,绘制家系系谱图,分析该家系患者临床特征和遗传表现。结果该家系共9人,其中DM1患者2例,存在遗传早现现象。2例DM1患者均有肌强直、肌无力等典型症状,伴有CNS、心脏、内分泌等多系统受累,EMG可见特征性大量肌强直电位出现,先证者行肌肉活检结果可见典型强直性肌营养不良伴镶边空泡病理改变,基因检测发现其DMPK基因均存在CTG三核苷酸大量重复扩增现象,给予奥卡西平治疗有效。结论DM1是以肌强直、肌无力为典型症状,伴有CNS、心脏、内分泌等多系统受累的遗传病,EMG、肌肉活检和基因检测可帮助确诊DM1,肌肉病理中伴镶边空泡少见,奥卡西平能改善肌强直症状。

抗CD3/CD20双特异性抗体生物学活性测定方法的建立

目的建立基于报告基因的抗CD3/CD20双特异性抗体生物学活性检测方法。方法以WIL2-S细胞系作为靶细胞,以Jurkat-NF-κB-Luc转基因细胞系作为效应细胞,对抗体的量效范围、孵育时间、诱导时间、效靶比及细胞接种密度进行优化,构建可用于测定抗CD3/CD20双特异性抗体生物学活性的报告基因法。依据ICHQ2的指导原则对方法进行全面验证,包括专属性、准确性、精密度、线性、稳定性。结果成功建立了具有量效关系的抗CD3/CD20双特异性抗体生物学活性检测方法,该方法的抗体起始浓度为500 ng/mL,稀释倍数为1∶4,共计8个浓度点(500,125,31.25,7.81,1.95,0.49,0.12,0.031 ng/mL),效靶比为4∶1,诱导时间为4 h。本方法具有良好的专属性;5个不同稀释组回收率样品经测定,相对效价分别为59.63%±4.57%,75.54%±4.05%,99.98%±9.63%,123.90%±5.54%和142.51%±12.82%;对应的回收率分别为93.17%±7.15%,94.42%±5.06%,99.98%±9.63%,99.12%±4.43%以及91.21%±8.21%,CV分别为7.67%,5.35%,9.63%,4.47%和9.00%,上述结果的变异系数CV值均<10%;实测效价与理论效价线性回归分析相关系数R^(2)=0.9823,线性良好;本研究建立的方法可以敏感检测出抗体结构变化对生物活性的影响,对双特异性抗体的稳定性检测有一定的指导意义。结论利用转基因细胞法成功建立了抗CD3/CD20双特异性抗体生物学活性检测方法,该方法专属性强、准确性高、重复性好,可用于评价抗CD3/CD20双特异性抗体生物学活性。

gltC基因缺失对单增李斯特菌10403S抗酸应激能力的影响

【目的】构建单增李斯特菌gltC基因缺失株,并阐明gltC基因对单增李斯特菌抗酸应激能力的影响。【方法】通过对单增李斯特菌参考菌株10403S进行添加或不添加外源性谷氨酸在致死性酸性条件的抗酸应激试验,测定谷氨酸对菌株抗酸应激存活能力的影响;利用同源重组方法构建单增李斯特菌gltC基因缺失株;通过生长曲线测定gltC基因缺失对菌株生长能力的影响;通过酸应激存活试验测定gltC基因缺失对单增李斯特菌酸应激能力的影响;通过Western blotting测定gltC基因缺失对GadD2蛋白表达水平的影响;通过构建携带gltBD启动子区域gfp报告质粒,测定在酸性和中性条件下gltC基因缺失对gltBD启动子区域转录水平的影响。【结果】酸应激结果显示,添加外源谷氨酸可极显著提高菌株10403S在pH 2.5酸性条件下的存活能力(P<0.01)。PCR结果显示,缺失株10403SΔgltC构建成功。生长曲线结果显示,gltC基因缺失不影响单增李斯特菌的生长能力。酸应激结果显示,在pH 2.5致死性酸性环境下缺失株10403SΔgltC生长能力弱于亲本株10403S(P<0.01)。Western blotting结果显示,gltC基因缺失不影响GadD2蛋白表达水平(P>0.05)。PCR结果显示,携带gltBD启动子区域的gfp报告质粒构建成功。GFP荧光分析结果显示,gltC基因缺失后在酸性和中性条件下均会极显著降低gltBD启动子区域转录水平(P<0.01)。【结论】gltC基因缺失不影响单增李斯特菌正常生长,缺失株10403SΔgltC表现出抗酸应激能力下降,且会降低gltBD启动子区域转录水平,但不影响GadD2蛋白表达水平。

基于颗粒酶B启动子的磁共振报告基因成像监测CAR-T细胞激活状态

目的利用颗粒酶B(granzyme B,GB)启动子控制铁蛋白(ferritin heavy chain,FTH1)报告基因表达,探讨通过磁共振成像(magnetic resonance imaging,MRI)监测嵌合抗原受体T细胞(chimeric antigen receptor T cells,CAR-T细胞)激活状态的可行性。方法通过Ficoll密度梯度离心法以及流式分选得到细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTLs)。将GB启动子和FTH1基因连接,连同二唾液酸神经节苷脂(disialoganglioside 2,GD2)嵌合抗原受体(chimeric antigen receptor,CAR)以慢病毒为载体转入CTLs,构建GD2-CAR-T/pGB-FTH1细胞,以GD2-CAR-T/pCMV-FTH1、GD2-CAR-T和T细胞为对照。CytoTox96@非放射性细胞毒性检测各组细胞与人神经母细胞瘤细胞(SK-N-SH)共培养后的杀伤效果,ELISA检测共孵育因子以及GB分泌量,Western blot、普鲁士蓝染色、细胞MRI检测共培养后FTH1基因的表达。结果成功获得CTLs并构建GD2-CAR-T/pGB-FTH1、GD2-CAR-T/pCMV-FTH1、GD2-CAR-T细胞,3组细胞对肿瘤细胞杀伤效果、共孵育因子以及GB分泌量均显著高于T细胞组,GB表达水平在与SK-N-SH细胞共培养后1 d最高,3 d和7 d依次降低。GD2-CAR-T/pGB-FTH1组FTH1相对表达量以及铁含量变化趋势与GB表达一致,MRI信号呈逐渐升高趋势。GD2-CAR-T/pCMV-FTH1组FTH1相对表达量、铁含量及MRI信号在各时间点均无显著差异。GD2-CAR-T和T细胞组无明显FTH1表达及聚铁效应。结论基于GB启动子的MRI报告基因成像可实时反映CAR-T细胞的GB表达水平和肿瘤杀伤作用,为CAR-T治疗提供了一种监测细胞激活状态的可视化手段。

散发先天性心脏病致病基因SOX7新突变的发现及功能研究

目的:寻找散发先天性心脏病(CHD)致病基因SOX7新突变并研究其功能。方法:选取116例散发CHD患儿和228名无CHD对照儿童,对其SOX7基因进行测序分析,以发现新的致CHD突变。克隆SOX7基因并构建野生型人SOX7表达质粒,通过定点诱变产生突变型人SOX7表达质粒,使用脂质体转染多种表达质粒入HeLa细胞,应用双报告基因定量分析突变的功能效应。结果:在1例男性散发先天性室间隔缺损患儿中发现了SOX7基因新突变,即NM_031439.4:c.361C>T;p.(Gln121*)突变;在其他115例CHD患儿和228名无CHD对照者中未检测出该突变。双报告基因定量研究表明,突变型人SOX7对其关键靶基因GATA4的转录激活作用消失。结论:SOX7基因突变可能是一部分散发CHD的病因,这对CHD的个体化防治有潜在的临床意义。

月季‘月月粉’与野蔷薇U6启动子的克隆及活性分析

【背景】U6启动子是CRISPR/Cas9基因编辑系统中驱动sgRNA转录的重要元件,而不同物种的U6启动子转录活性可能存在差异,且物种内源U6启动子相比物种外源U6启动子可能具有更高效的启动效率。迄今对蔷薇属(Rosa)植物的U6启动子少有报道,且尚未获得转录活性强于拟南芥U6的启动子。【目的】筛选转录活性更高的蔷薇属植物U6启动子,为后续月季和野蔷薇等蔷薇属植物CRISPR/Cas9基因编辑系统的优化和分子育种奠定基础。【方法】利用拟南芥保守的102 bp U6 snRNA序列,从中国古老月季‘月月粉’(Rosa chinensis Old Blush,OB)基因组中克隆相似性最高的6个OBU6启动子,从野蔷薇(R.multiflora)基因组中克隆相似性最高的7个RmU6启动子,并构建OBU6启动子和RmU6启动子分别驱动LUC和GUS报告基因的融合表达载体,利用农杆菌介导的瞬时转化法转染本氏烟草(Nicotiana benthamiana)叶片和月季‘萨曼莎’(R.Samantha)组培苗,通过双荧光素酶活性检测和GUS组织化学染色对月季和野蔷薇的启动子转录活性分别进行比较。【结果】‘月月粉’6个OBU6启动子和野蔷薇7个RmU6启动子均具有影响U6启动子转录活性的两个必要元件USE和TATA box。双荧光素酶活性检测和GUS组织化学染色结果显示,月季‘月月粉’OBU6启动子和野蔷薇RmU6启动子均具有转录活性,但OBU6启动子的转录活性均弱于拟南芥AtU6-1启动子。考虑到过长的U6启动子可能会削弱其转录活性,于是选取其中转录活性相对较高的OBU6-4进行5′端截短(1507、1076、574和187 bp),但截短后的启动子未能提高转录活性;而在野蔷薇中,成功筛选到一个长度为630 bp的RmU6-2启动子,其转录活性显著高于拟南芥AtU6-1启动子。【结论】从月季‘月月粉’中克隆得到6条U6启动子,从野蔷薇中克隆得到7条U6启动子,并筛选出一条转录活性显著高于拟南芥AtU6-1启动子的野蔷薇U6启动子RmU6-2,为构建蔷薇属植物CRISPR/Cas9基因组编辑系统提供了极具应用潜力的启动子。

伴有ABCA1基因突变的青年代谢相关脂肪性肝硬化1例报道

代谢相关脂肪性肝病(metabolic dysfunction-associated fatty liver disease,MAFLD)是遗传易感个体长期营养过剩导致的慢性代谢应激性肝损伤,其不良结局为肝硬化、肝细胞癌和肝功能衰竭[1,2]。本文报道1例28岁年轻男性在腹腔镜下胆囊切除术时发现肝硬化改变,遂在我院经基因检测和肝穿刺活检确诊为伴有ABCA1基因突变的MAFLD导致的肝硬化。现回顾性分析该病例的诊疗过程并进行文献复习,供临床医生参考。

转录因子NF-κB对鲤疱疹病毒Ⅱ型基因启动子的表达调控研究

为深入探究NF-κB信号通路对CyHV-2感染过程的影响,在CyHV-2感染的GiCF细胞中加入NF-κB激动剂,通过RT-qPCR和Western Blot分析在感染24、72、96 h后CyHV-2编码基因在转录和翻译水平的表达情况。结果显示,在加入NF-κB激动剂后的72、96 h,CyHV-2编码基因转录和翻译水平均明显高于对照组,提示NF-κB促进CyHV-2编码基因的转录及翻译。为进一步阐明NF-κB如何影响CyHV-2的转录和翻译,利用生物信息学软件预测CyHV-2部分编码基因的启动子序列中和NF-κB的结合位点,采用双荧光素酶报告基因实验分析NF-κB对启动子活性的影响。结果表明,NF-κB过表达提高了病毒基因启动子活性,提示NF-κB可以通过促进CyHV-2基因的启动子活性来增强病毒复制水平。研究结果有助深入探究CyHV-2基因的表达翻译机制,也为开发基于NF-κB信号通路靶点的新的抗病毒抑制剂提供理论支撑。