双报告基因真核表达载体的构建及其功能鉴定

目的:构建绿色荧光蛋白和海肾荧光素酶共同高效表达的双报告基因真核表达载体。方法:将增强型绿色荧光蛋白基因和海肾荧光素酶基因以昆虫病毒T2A序列相连接而后克隆进入pcDNA3.1(-)质粒,构建双报告基因真核表达载体。将该载体转染至COS-7细胞,通过荧光显微镜观察、照度计定量分析检测绿色荧光蛋白和海肾荧光素酶生物活性,Western Bolt检测T2A序列自剪切效率。结果:双报告基因真核表达载体能够同时表达非融合的绿色荧光蛋白和海肾荧光素酶,与单独表达载体产物具有相似的生物活性和表达效率。结论:双报告基因真核表达载体建立成功,为基因表达调控等相关领域研究提供辅助工具。     

A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)

A construct suitable for genome-wide transfer-DNA (T-DNA) and subsequent transposon-based (Ds) gene trapping has been developed for use in rice (Oryza sativa). This T-DNA/Ds construct contains: Ds terminal sequences immediately inside T-DNA borders for subsequent Ds mobilization; promoterless green fluorescent protein (sgfpS65T) and beta-glucuronidase (uidA) reporter genes, each fused to an intron (from Arabidopsis GPA1 gene) to enable bidirectional gene trapping by T-DNA or Ds; an ampicillin resistance gene (bla) and a bacterial origin of replication (ori) to serve as the plasmid rescue system; an intron-containing hygromycin phosphotransferase gene (hph) as a selectable marker or Ds tracer; and an intron-containing barnase gene in the binary vector backbone (VB) to select against transformants carrying unwanted VB sequences. More than a threefold increase over previously reported reporter gene-based gene trapping efficiencies was observed in primary T-DNA/Ds transformant rice lines, returning an overall reporter gene expression frequency of 23%. Of the plant organs tested, 3.3-7.4% expressed either reporter at varying degrees of organ or tissue specificity. Approximately 70% of the right border (RB) flanking sequence tags (FSTs) retained 1-6 bp of the RB repeat and 30% of the left border (LB) FSTs retained 5-23 bp of the LB repeat. The remaining FSTs carried deletions of 2-84 bp inside the RB or 1-97 bp inside the LB. Transposition of Ds from the original T-DNA was evident in T-DNA/Ds callus lines super-transformed with a transposase gene (Ac) construct, as indicated by gene trap reporter activity and rescue of new FSTs in the resulting double transformant lines.     

徐州医学院病原生物学与免疫学教研室

目的:建立高活性的PSA启动子荧光素酶报告载体。方法:提取前列腺癌细胞PC-3总DNA,PCR扩增出PSA启动子片段,建立PSA启动子荧光素酶重组载体PGL3-psap,通过脂质体介导将重组载体转染到前列腺癌细胞PC-3中,使用荧光素酶检测系统检测其活性。结果:DNA测序结果表明成功构建荧光素酶重组载体PGL3-psap,荧光素酶检测显示重组载体在前列腺癌细胞PC-3中有较强活性。结论:本研究成功构建了具有高度活性的PSA启动子荧光素酶报告载体,为进一步研究前列腺癌的诊断和治疗提供了实验基础。     

大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体的构建

目的构建大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体,并检测其在神经元中的表达。方法采用PCR方法获得GluR2基因启动子区目的片段（-298～＋283）,双酶切后插入到pGL3-Basic载体中构成重组表达载体,使萤火虫荧光素酶报告基因的表达受GluR2启动子控制。将构建的重组表达载体或pGL3-Basic载体分别与内参质粒pRL—CMV（表达海肾荧光素酶）共转染原代培养皮质神经元,24h后用双荧光检测试剂盒测定萤火虫荧光素酶及海肾荧光素酶活性。结果重组表达载体经双酶切及测序鉴定证明构建正确,该重组表达载体在神经元中特异性高表达萤火虫荧光素酶。结论成功构建大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体,并检测到该载体在神经元中的特异性表达。     

Connexin43 mRNA contains a functional internal ribosome entry site

A reporter gene construct was used to study the regulation of connexin43 (Cx43) expression, the major gap junction protein found in heart and uterus, in transfected cell lines. The construct had the firefly luciferase gene under the control of the Cx43 promoter. Inclusion of the 5'-untranslated region (UTR) of the mRNA in the construct increased luciferase expression by 70%. A bicistronic vector assay demonstrated that the Cx43 5'-UTR contains a strong internal ribosome entry site (IRES). Deletion analysis localized the IRES element to the upstream portion of the 5'-UTR.     

A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors

Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively.     

p53 promoter-based reporter gene in vitro assays for quick assessment of agents with genotoxic potential

The p53 promoter-based green fluorescent protein(GFP)and luciferase reporter gene assayshave been established for detecting DNA damage induced by genotoxic agents.To evaluate the system,NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or5-fluorouracil.Expression of the GFP reporter gene was significantly and specifically induced in the cellsexposed to mitomycin or 5-fluorouracil.Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin,5-fluorouracil or cisplatin at various concentrations.Similarly,exposure of thecells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reportergene expression.Thus,these in vitro reporter gene assays could provide an ideal system for quick assess-ment or screening of agents with genotoxic potential.     

高效缺氧诱导表达载体的构建与鉴定

目的：构建缺氧诱导表达载体,以介导报告基因在缺氧环境下的特异、高效表达。方法：通过分子生物学方法,将鼠磷酸甘油酸激酶基因的缺氧应答元件（HRE）和最小CMV（mCMV）启动子重组,构建增强型绿色荧光蛋白（EGFP）或萤光素酶报告基因的可诱导载体;通过酶切鉴定和测序分析,证实载体获得正确的构建;将重组载体转染HeLa细胞,观察EGFP荧光强度并检测萤光素酶活性。结果：HRE／mCMV启动子调控的报告基因载体具有特异和高效的诱导活性。结论：构建了可以进行特异和高效缺氧诱导的报告基因载体,为其进一步的开发和应用奠定了实验基础。     

SARS冠状病毒N蛋白激活hfgl2凝血酶原酶基因的研究

研究鉴定激活hfgl2凝血酶原酶基因的SARS冠状病毒结构蛋白。从SARS尸检肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、S2和M全长基因序列,再分别克隆到真核表达载体pcDNA3.1( )上。应用免疫组织化学分析鉴定pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2的表达。构建人纤维介素(hfgl2)启动子荧光素酶报告基因质粒,并将SARS冠状病毒结构蛋白表达质粒分别与其共转染以明确激活hfgl2基因转录的SARS冠状病毒结构蛋白。将目的片段克隆至pcDNA3.1( ),经酶切鉴定和测序鉴定无误;免疫组织化学染色可见明显的CHO细胞胞浆棕染。与hfgl2启动子共转染实验阐明SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高4.6倍。SARS冠状病毒N蛋白可增强hfgl2基因的转录活性。     

血管内皮生长因子基因3'UTR 不同基因型载体的构建与鉴定

目的:构建含SNP位点的血管内皮生长因子(VEGF)基因3’UTR的荧光素酶报告基因载体,为进一步揭示VEGF基因3’UTR的单核苷酸多态性(SNP)影响肺癌发病风险的分子机制奠定基础。方法:以rs3025039和rs3025040两个位点均为C纯合子的非癌症病人血液DNA为模板,扩增出两位点为C/C单体型、长度为1448 bp的VEGF基因3’UTR目的片段,测序验证后将其克隆至pMIR-REPORT荧光素酶报告基因载体上,得到重组质粒pMIR-C/C。同时,我们以pMIR-C/C为模板定点突变两个SNP位点,得到具有T/T单体型的重组质粒pMIR-T/T。将各重组质粒转化大肠杆菌DH10B,筛选阳性克隆后提取质粒进行双酶切鉴定及DNA测序鉴定。结果:单菌落质粒测序验证显示带有C/C单体型的VEGF基因3’UTR重组质粒pMIR-C/C构建成功;经两次定点突变,成功将pMIR-C/C质粒转变为pMIR-T/T,经测序验证未引入任何其他突变。同时生物信息学预测还显示rs3025040位点位于miR-199a/b与VEGF基因mRNA的结合位置,其改变可以影响miRNA与mRNA的结合效率。结论:本研究成功构建了含有两个连锁SNP的VEGF基因3’UTR的荧光素酶报告基因载体,为今后VEGF基因3’UTR的功能研究奠定基础。     

稳定表达大鼠GCLC启动子及荧光素酶报告基因细胞株的建立

目的:建立稳定表达大鼠谷氨酰半胱氨酸合成酶催化亚单位(GCLC)启动子及荧光素酶报告基因的大鼠肺泡上皮细胞株。方法:克隆大鼠GCLC上游5.9kb的启动子序列并构建重组报道载体PGL4.19-GCLC-LUC,转染到大鼠Ⅱ型肺泡上皮细胞L2,经G418筛选以获得单细胞抗性克隆并在传代过程中能稳定表达荧光素酶活性;检测细胞荧光素酶表达与细胞数量相关性;PCR检测稳定细胞株基因组已整合的插入片段;刺激因子刺激6h,检测稳定细胞株的反应性。结果:成功构建PGL4.19-GCLC-LUC;构建的稳定细胞株能稳定地表达荧光素酶,且与细胞数量正相关;PCR检测稳定细胞株目的片段稳定整合基因组;TNF-α刺激后,能使荧光素酶活性上升,差异有统计学意义(P0.05);Rapamycin,GSH-EE刺激后,荧光素酶活性显著下降(P0.05)。结论:成功构建稳定表达大鼠GCLC启动子及荧光素酶报告基因的细胞株,将为高通量药物筛选以及进一步研究GCLC基因的转录调控提供重要的细胞研究手段。     

Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress

Background  

Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc ⁺) gene and low temperature-responsive luciferase activation was monitored in real time.     

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.     

核酸疫苗双表达载体的构建及表达

将微小病毒内部核糖体进入位点(IRES)基因克隆到质粒pVAXI载体多克隆位点,构建出核酸疫苗双表达载体pVI。将绿色荧光蛋白(EGFP)基因和新霉素磷酸转移酶(neor)基因作为报告基因,连接到pVI载体IRES基因的前后两处多克隆位点,构建出表达载体pEIN。通过脂质体介导的方法将该载体转染COS-7细胞,筛选到同时表达绿色荧光蛋白和新霉素磷酸转移酶的表达株,表明成功地构建了核酸疫苗双表达载体,为构建多价核酸疫苗及带有分子佐剂的核酸疫苗打下了基础。     

Mir-106b对阿尔茨海默病昼夜节律调控的初步研究

目的探讨mir-106b在阿尔茨海默病发病中的作用。方法取6月龄APPswe/PSΔE9小鼠脑组织,进行microRNA芯片的检测;利用real-time PCR对芯片检测结果进行验证;构建mir-106b表达载体,将其转染至SH-SY5Y细胞中构建mir-106b稳定转染的稳转细胞系。用Targetsan、Pictar、miRanda等靶基因预测软件,对mir-106b的靶基因进行预测,根据靶基因的功能选择可能与AD发病相关的靶基因,并用Western blot对所选择的靶基因在稳转细胞中的表达进行验证。用双荧光素酶报告检测系统检测mir-106b与其靶基因的结合位点。结果芯片结果显示,与野生型小鼠相比,6月龄APPswe/PSΔE9小鼠脑组织中mir-106b的表达下降,经real-time PCR验证,mir-106b的表达在APPswe/PSΔE9小鼠脑组织中的表达较野生型小鼠升高,差别具有统计学意义（P=0.03）。mir-106b稳转细胞系较对照mir-106b的表达升高2～5倍。神经PAS结构域蛋白2（neuronal PAS domain protein2,NPAS2）在mir-106b稳转细胞中的表达明显下降。双荧光素酶报告实验证实：与对照相比,带有NPAS2的3＇UTR的荧光素酶报告载体与mir-106b表达载体共转染,海肾荧光素酶/萤火虫荧光素酶的相对活性降低;荧光素酶报告载体的NPAS2-3＇UTR突变后与mir-106b表达载体共转染,海肾荧光素酶/萤火虫荧光素酶的相对活性升高。结论mir-106b可能通过调节机体生物钟基因NPAS2的表达,影响阿尔茨海默病人的生活节律,参与阿尔茨海默病的发生。