基因,报告

摘要： 目的 建立检测聚乙二醇化胰高血糖素样肽1类似物(polyethylene glycolylated glucagon-like peptide-1 analogue,PEG-GLP-1a)生物活性的报告基因法,并对该方法进行验证.方法 将人胰高血糖素样肽1受体表达载体和分泌型碱性磷酸酶(secreted alkaline phosphatase,SEAP)报告基因载体瞬时共转染HEK293T细胞,培养后加入PEG-GLP-1a刺激细胞分泌SEAP,使用SEAP报告基因检测试剂盒进行测定,并验证其专属性、准确性、精密度、稳定性等指标.结果 人胰高血糖素样肽1受体和SEAP双载体瞬时共转染的HEK293T细胞对PEG-GLP-1a具有强反应性.使用该方法测定PEG-GLP-1a具有良好的专属性.加标回收率为97.6％～102.2％,准确性较好.不同测定日期的日内半数效应浓度的变异系数均不超过10.22％,不同测定日期的日间半数效应浓度的变异系数均不超过13.02％.结论 报告基因法可以用于PEG-GLP-1a的生物活性检测.

摘要： As an important in vivo noninvasive molecular imaging modality, PET imaging can quantify and visualize the serial trafficking, tumor targeting, cell number maintenance, cell expansion, activation and immunological function of adoptive immune cells in cancer cell therapy. Thereby it may play a significant role in the treatment options and efficacy evaluation for cellular immunotherapy in cancer. This review focuses on PET reporter gene imaging which has been studied intensively and applied widely in the research on imaging monitoring of cellular immunotherapy for cancer, with the purpose to provide innovative clues for the preclinical study and clinical translation research.%作为一种重要的在体无创分子影像手段,PET显像能够实现过继免疫细胞的在体运输、肿瘤靶向、数量维持、扩增活化及免疫功能等方面的定量可视化监测,从而在肿瘤的细胞免疫治疗方案选择及临床疗效评估中发挥重要作用.笔者就目前研究较深入、应用较为广泛的肿瘤细胞免疫治疗的PET报告基因显像进行重点阐述,旨在为日后进一步的临床前研究及临床转化应用提供新思路.

摘要： Objective To explore the effect of microRNA-203(miR-203) on the expression of collagen type Ⅳ alpha 4(COL4A4) and its role in oral submucous fibrosis(OSF).Methods It has been revealed that COL4A4 is the putative target of miR-203 by retrieving a variety of bioinformatics software tools.The expression of COL4A4 protein in the indicated tissues was examined by Western blotting analysis and the potential binding sites of miR-203 and COL4A4-3'UTR was analyzed by using bioinformatics.This study integrated a fragment of COL4A4 3'UTR containing the target sequence into the pMIR-REPORT luciferase vector.Then,the COL4A4 3'UTR-luciferase and miR-203 were contransfected into 293T cells.Beta-gal was used as a control to monitor transfection efficiency.Cells were harvested and luciferase activity was measured after 24 h of transfection.Results The relative expression mean valves of COL4A4 were 2.46±2.26 in OSF and 0.70±0.49 in normal control.The expression of COL4A4 in OSF was higher than normal control(P＜0.05) and the difference between groups was statistically significant.The relative expression mean valves of miR-203 were 2.57±2.09 in OSF and 154.07±191.11 in normal control.The expression of miR-203 in OSF was lower than in normal control(P＜0.01) and the difference between groups was statistically significant.Bioinformatics analysis revealed that the COL4A4 3'UTR had a conserved site (site 1) and 3 non-conserved sites(site 2,site 3,site4) complementary to the miR-203.MiR-203 could significantly inhibit site 1-luciferase reporter activity,partially inhibited site 3-and site 4-1uciferase reporter activities,while no significant effect on site 3-luciferase reporter activity.Conclusions MiR-203 was negatively correlated with the expression of COL4A4 protein in OSF.Moreover,miR-203 repressed the expression of COL4A4 by targeting 3'UTR site of COL4A4 mRNA.%目的 构建微小RNA-203(microRNA-203,miR-203)表达质粒和Ⅳ型胶原蛋白α4链(collagen typeⅣalpha 4,COL4A4) 3'UTR荧光素酶报告基因,探究miR-203对COL4A4的靶向调控作用及其在口腔黏膜下纤维化(oral submucous fibrosis,OSF)中的作用.方法 通过生物信息学软件对miR-203可能调控的靶基因进行预测,结合其生物学功能,筛选出miR-203可能的靶基因COL4A4.收集2016年1至4月在中南大学湘雅口腔医院黏膜科就诊、具有典型病理特征、未经治疗、不伴其他口腔疾病的9例OSF患者颊黏膜组织(OSF组)及其配对的正常组织(对照组),应用蛋白质印迹法检测两组标本中COL4A4的表达,实时定量聚合酶链反应法检测miR-203的相对表达量.通过生物信息学软件(http://www.targetscan.org/)寻找COL4A4基因3UTR与miR-203之间的作用位点.将COL4A4基因3 UTR包括miR-203结合位点、上下游部分侧翼序列以及酶切位点在内共60 bp片段分别克隆入哺乳动物细胞miRNA表达报告载体(pMIR-REPORT Luciferase)荧光素酶报告基因载体,将其分别与β-半乳糖苷酶表达质粒及miR-203表达载体共转染293T细胞,24 h后检测报告基因活性.结果 COL4A4的相对表达水平OSF组(2.46±2.26)显著高于对照组(0.70±0.49)(P＜0.05);miR-203的相对表达水平(2-△△CT) OSF组(2.57±2.09)显著低于对照组(154.07±191.11)(P＜0.01).生物信息预测显示,COL4A4基因3'UTR与miR-203之间有一物种间保守作用位点(位点1),3个非保守作用位点(位点2,3,4);miR-203能明显抑制位点1介导的报告基因活性,能部分抑制位点2与位点4介导的报告基因活性,而对位点3介导的报告基因活性无显著影响.结论 在OSF组织中,miR-203与COL4A4的蛋白表达呈负相关,miR-203靶向结合COL4A4基因3'UTR,抑制COL4A4蛋白表达.

摘要： 目的 构建靶向CEA的以Ⅰ型内含子核酶为载体核心介导的生物发光-核素双报告基因显像系统.方法 采用基因工程技术构建靶向CEA的Ⅰ型内含子核酶及核酶-荧光素酶-胸苷激酶(Rib53-Fluc-tk)报告质粒,采用细胞生物发光等方法检测核酶反式剪接产物的表达;以Iodogen固相氧化法合成131I-氟-碘阿糖呋喃基尿嘧啶(FIAU),行细胞摄取实验.采用两样本t检验、方差分析及最小显著差异(LSD)t检验进行统计分析.结果 Rib53-Fluc-tk测序正确;乳腺癌细胞MCF-7的荧光素酶比值增高,可达0.64±0.10(n=4);MCF-7细胞转染Rib53-Fluc-tk后的反式剪接产物条带大小为520 bp,测序结果正确;pCDNA3.1-CEA及Rib53-Fluc-tk共转人胚肾细胞293T,可探测到生物发光信号.131 I-FIAU的标记率为(64.02±4.79)％(n=3),放化纯为(95.96±1.07)％(n=3),标记产物在人血清中24h的稳定性高.293T单独转染Rib53-Fluc-tk,细胞摄取率仅为(0.31±0.01)％(n=4);293T共转染pCDNA3.1-CEA、Rib53-Fluc-tk质粒,细胞摄取率增加至(1.40±0.06)％(n=4),F=1 007.29,t=136.34,P＜0.01.结论 成功构建了Ⅰ型内含子核酶为核心介导的靶向CEA的双报告基因系统,并对CEA mRNA进行了生物发光以及细胞摄取的检测,为改善传统报告基因显像的特异性奠定了体外实验的基础.%Objective To develop a specific trans-splicing intron ribozyme type Ⅰ-mediated dual reporter gene system (Rib53-Fluc-tk) for targeting CEA.Methods The novel CEA-targeting trans-splicing ribozyme with the downstream reporter system (Rib53-Fluc-tk) was constructed by genetic engineering technology.The trans-splicing reaction product was evaluated using the 131I-5-iodo-2'-fluro-l-beta-D-arabinofuranosy-luracil (FIAU) cellular uptake rates and the bioluminescence.Two-sample t test,the analysis of variance and the least significant difference (LSD) t test was performed for data analysis.Results The sequence of Rib53-Fluc-tk was proved by gene-sequencing test.Human MCF-7 breast cancer cells showed a high ratio of firefly luciferase/renilla luciferase (0.64±0.10,n =4).A 520 bp band of product existed,which matched with the predicted size using RNA from cells transfected with Rib53-Fluc-tk in MCF-7.Signals were detected by bioluminescence in human embryonic kidney 293T cells co-transfected with Rib53-Fluc-tk and pCDNA3.1-CEA.The labelling rate of 131I-FIAU was (64.02±4.79)％ (n =3).The radiochemical purity was (95.96± 1.07)％ (n=3),and the stability of the radiocompound remained high in human serum at least for 24 h.The uptake of 131I-FIAU in 293T cells transfected with Rib53-Fluc-tk was (0.31±0.01)％ (n=4),while it increased with the incubation time in 293T cells co-transfected with pCDNA3.1-CEA and Rib53-Fluc-tk and reached (1.40±0.06)％ at 4.5 h (F=1 007.29,t=136.34,both P＜0.01).Conelusions A novel and specific reporter gene in the cellular level was established.Taking advantage of trans-splicing reaction of the ribozyme,it could improve the specificity of the reporter gene imaging.

摘要： Objective To explore the specificity and efficiency of YFP labeled natural killer (NK) cells through Vav-Cre induced YFP reporter system in mice. Methods ROSA26R-YFP and Vav-Cre mice were crossed, and their YFP and Cre gene double positive progeny were screened by genotyping. The specificity of YFP in hematopoietic cells from im-mune organs including lymph nodes, spleen, thymus and bone marrow were analyzed by flow cytometry. The percentages of YFP positive cells in NK cells from lymph nodes, spleen and bone marrow were also analyzed by flow cytometry. Results A total of 11 double positive mice (ROSA26R-YFP-(+/-)VavCre) were obtained in 17 mouse offspring by crossing ROSA26R-YFP mice with Vav-Cre mice. The percentages of YFP positive cells in immune organs including lymph nodes, spleen, thy-mus and bone marrow were 73.87%± 1.51%, 56.07%± 1.47%, 86.17%± 1.74%and 53.60%± 3.56%, and there were signifi-cant differences compared with the corresponding negative control cells(0.27%±0.01%, 1.33%±0.91%, 0.11%±0.01%and 0.29%± 0.03%, P0.05). The positive rates of YFP were significantly higher in NK cells in lymph nodes, spleen and bone marrow (76.94%±0.84%、81.66%±1.18%and 88.92%±0.77%) compared with those of control (P0.05）；淋巴结、脾和骨髓中NK细胞YFP阳性百分比（%）76.94±0.84、81.66±1.18、88.92±0.77，与阴性对照组比较均明显增高（均P<0.01）。结论 Vav-Cre介导的YFP报告基因系统标记小鼠体内NK细胞具有特异性及高效性。

摘要： 目的 制备含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的辛德毕斯病毒,并对其进行鉴定.方法 采用融合PCR技术将报告基因EGFP融合到辛德毕斯病毒XJ-160感染性克隆pBR-XJ160的结构基因编码框后,并在报告基因前面添加亚基因启动子SP6,通过反向遗传学技术拯救得到EGFP标记的辛德毕斯病毒.结果 成功拯救并获得了EGFP标记的XJ-160病毒,该重组病毒具有良好的荧光表达特性和遗传稳定性.结论 EGFP标记的辛德毕斯病毒可用于观测病毒的侵染轨迹,为进一步研究辛德毕斯病毒嗜性、生物学功能,以及感染机制奠定了基础.%Objective To construct and characterize EGFP reporter gene labeled Sindbis virus (SINV).Methods The reporter gene EGFP was inserted into the genome of infectious clone pBR-XJ160 by using multi-fusion long fragment PCR method.Then apply reverse genetic manipulation technique to rescue and obtain EGFP labeled SINV.Results We successively obtained labeled SINV,which has good fluorescent expression characteristics and genetic stability.Conclusion The labeled virus can be seen in living cells and living body,and this serves as a good tool for cell and tissue tropism and biological function study of viruses.This study laid a foundation for further studying the cell tropism,biological functions and infection mechanism of SINV.

摘要： 目的 建立小鼠双特异性磷酸酶1(DUSPl)3’端非翻译区(UTR)双荧光素酶报告基因表达系统,并对其进行功能鉴定.方法 提取RAW264.7细胞总RNA并反转录为cDNA,以其为模板通过PCR扩增获得小鼠DUSP1 3'-UTR全长序列,酶切后克隆至pGL3-control载体荧光素酶编码基因的下游,获得重组载体LuDP,经PCR、限制性内切酶酶切、DNA序列分析鉴定正确后与内参照质粒pRL-TK共转染NIH3T3细胞,48h后检测双荧光素酶报告基因的表达.将含有小鼠DUSP1 3’-UTR的荧光素酶表达模块与来源于pRL-TK质粒的RL表达模块克隆至pLenti6-TR质粒中,获得双荧光素酶报告基因表达载体LuDP/RL,将其与RNA结合蛋白HuR表达载体(pcDNA3.1-HuR-FLAG)、mmu-miR-101a分别共t转染NIH3T3细胞,检测双荧光素酶的活性,分析HuR、mmu-miR-101对荧光素酶基因表达活性的影响.结果 成功构建了双荧光素酶报告基因表达载体.小鼠DUSP1 mRNA 3'-UTR可显著下调与其相连的荧光素酶的表达活性(0.14±0.01倍,P＜0.01)；共转染HuR可上调荧光素酶的表达活性(1.40±0.20倍,P＜0.05),共转染mmu-miR-101a则可下调荧光素酶表达活性(0.57±0.18倍,P＜0.05).结论 成功构建了DUSP1 3’-UTR双荧光素酶报告基因表达系统,该系统可用于转录后调控元件对小鼠DUSP1基因表达的调控机制研究.%Objective To construct mouse dual specificity phosphatase-1 (DUSP1) 3'-untranslated region (3'-UTR) dual luciferase reporter system, and to identify its function. Methods The total RNA of RAW264.7 murine macrophage cells was extracted and reverse-transcribed into cDNA. The full length of mouse DUSPl 3'-UTR fragment was amplified by PCR and sub-cloned to the immediate downstream of luciferase cDNA in pGL3-control luciferase expression plasmid to obtain the recombinant plasmid pGL3-Luc-DUSPl-3'UTR (LuDP). LuDP was then verified by PCR, restriction endonuclease and DNA sequencing analysis. Plasmid LuDP was transiently co-transfected into NIH3T3 cells with internal control plasmid pRL-TK, and the effects of DUSPl 3'-UTR on the expression of attached luciferase gene was analyzed with dual luciferase reporter assay system 48 hours after transfection. Both the luciferase expression module from LuDP and renilla luciferase expression module from pRL-TK were co-subcloned into pLenti6-TR vector to construct dual luciferase reporter plasmid LuDP/RL. The recombinant plasmid LuDP/RL was co-transfected with pcDNA3.1-HuR-FLAG (the eukaryotic expression plasmid of binding protein HuR) and miRNA mimics mmu-miR-101a into NIH3T3 cells respectively. The effects of HuR and mmu-mi-R101a on the expression of DUSPl 3'-UTR attached luciferase gene were also analyzed with dual luciferase reporter assay system. Results The dual luciferase reporter plasmid LuDP/RL was successfully constructed. The mouse DUSPl mRNA 3'-UTR significantly down-regulated the expression of attached luciferase gene in NIH3T3 cells (0.14 ± 0.01 fold, P<0.0l). The co-transfected HuR up-regulated the expression of luciferase in LuDP/RL plasmid (1.40 ± 0.20 fold, P<0.05), and co-transfected mmu-miR-101a down-regulated the expression of LuDP/RL (0.57 ±0.18 fold, P<0.05). Conclusion The constructed dual luriferase reporter plasmid LuDP/RL can be used to analyze the regulatory mechanism of mouse DUSP1 gene expression controlled by post-transcriptional regulatory elements.

摘要： 目的 构建携带萤火虫荧光素酶(Luc)报告基因的腺病毒载体(Ad-Luc),研究其感染大鼠骨髓间充质干细胞(BMSC)后的体内外生物发光成像.方法 从psiCHECK-2质粒中用PCR扩增Luc基因,克隆入腺病毒穿梭载体pShuttle-CMV后行Nhe Ⅰ/Xba Ⅰ双酶切和测序鉴定.重组腺病毒穿梭载体与骨架载体pAdeno同源重组并包装纯化后,测定其病毒滴度.用重组Ad-Luc感染BMSC,行体外生物发光成像确定最佳感染复数(MOI),并采用曲线拟合回归分析生物发光强度与MOI的关系.以锥虫蓝染色法评价细胞活力变化,计算细胞存活率.将转染后BMSC(1×106个)植入SD大鼠前肢肌肉内,行体内生物发光成像.细胞存活率组间比较采用两因素重复测量资料方差分析.结果 经酶切和测序鉴定证明,Ad-Luc构建成功,病毒滴度为1×1010空斑形成单位(PFU)/ml.体外生物发光检测结果显示最佳MOI值为50,Ad-Luc可高效感染BMSC,使其表达Luc,且拟合曲线示细胞生物发光强度随MOI增加而增强(R2 =0.98).转染组和未转染组细胞培养1、3、5、7d时,细胞存活率分别为(92.5±2.3)％与(94.1±1.8)％、(91.4±0.9)％与(92.7±2.0)％、(92.1±1.6)％与(93.3±2.4)％、(91.9±1.5)％与(93.0±3.1)％,2组间细胞活力的差异无统计学意义(F=4.38,P＞0.05).体内生物发光成像结果示BMSC移植1、3、7d后仍有存活,但随时间延长,生物发光信号逐渐减弱.结论 Luc报告基因通过腺病毒载体成功转入BMSC,实现了光学报告基因成像对移植干细胞的示踪.%Objective To construct adenovirus vector containing firefly luciferase reporter gene (AdLuc) and infect bone marrow mesenchymal stem cells (BMSC),then to take bioluminescence imaging in vitro and in vivo for identification.Methods The luciferase gene was amplified with PCR from psiCHECK-2 plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV).It was confirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing.PShuttle-CMV-Luc and backbone vector (pAdeno) were homologous recombined.Then the recombinant plasmid was packaged in HEK293 cells and the virus titer was detected.The BMSC were infected by the recombinant adenovirus.The bioluminescence imaging in vitro was performed to determine the best multiplicity of infection (MOI),and the relationship between bioluminescence intensity and MOI was analyzed by curve fitting regression analysis.Viability was evaluated via Trypan blue staining.The transfected BMSC (l× 106) were implanted into the muscles of forelimb of SD rats,and then tracked by bioluminescence imaging in vivo.Cell viability was compared using two-way repeated measures analysis of variance between groups.Results Enzyme digestion and sequence analysis indicated that Ad-Luc was successfully constructed.The virus titer was 1 × 1010 plaque forming unit (PFU)/ml.The bioluminescence detection in vitro showed that Ad-Luc could infect BMSC high efficiently to express luciferase and the best MOI was 50.The bioluminescence intensity enhanced with increase of MOI (R2 =0.98).No statistically significant difference was found in cell viability between transfected and untransfected BMSC at 1,3,5,7 d.The cell survival rates were (92.5±2.3)％ vs (94.1±1.8)％,(91.4±0.9)％ vs (92.7±2.0)％,(92.1±1.6)％ vs (93.3± 2.4) ％,(91.9 ± 1.5) ％ vs (93.0 ± 3.1) ％,respectively (F =4.38,P ＞ 0.05).The bioluminescence imaging in vivo showed that BMSC survived 1,3,7 d after implantation.However,bioluminescence signal decreased gradually over time.Conclusion It is feasible to apply the optical reporter gene imaging for tracing transplanted stem cells in vitro and in vivo due to the effective transformation of luciferase reporter gene into BMSC by adenovirus vector.

摘要： 目的 构建携带萤火虫荧光素酶(Luc)报告基因的腺病毒载体(Ad-Luc),研究其感染大鼠骨髓间充质干细胞(BMSC)后的体内外生物发光成像.方法 从psiCHECK-2质粒中用PCR扩增Luc基因,克隆入腺病毒穿梭载体pShuttle-CMV后行Nhe Ⅰ/Xba Ⅰ双酶切和测序鉴定.重组腺病毒穿梭载体与骨架载体pAdeno同源重组并包装纯化后,测定其病毒滴度.用重组Ad-Luc感染BMSC,行体外生物发光成像确定最佳感染复数(MOI),并采用曲线拟合回归分析生物发光强度与MOI的关系.以锥虫蓝染色法评价细胞活力变化,计算细胞存活率.将转染后BMSC(1×106个)植入SD大鼠前肢肌肉内,行体内生物发光成像.细胞存活率组间比较采用两因素重复测量资料方差分析.结果 经酶切和测序鉴定证明,Ad-Luc构建成功,病毒滴度为1×1010空斑形成单位(PFU)/ml.体外生物发光检测结果显示最佳MOI值为50,Ad-Luc可高效感染BMSC,使其表达Luc,且拟合曲线示细胞生物发光强度随MOI增加而增强(R2 =0.98).转染组和未转染组细胞培养1、3、5、7d时,细胞存活率分别为(92.5±2.3)％与(94.1±1.8)％、(91.4±0.9)％与(92.7±2.0)％、(92.1±1.6)％与(93.3±2.4)％、(91.9±1.5)％与(93.0±3.1)％,2组间细胞活力的差异无统计学意义(F=4.38,P＞0.05).体内生物发光成像结果示BMSC移植1、3、7d后仍有存活,但随时间延长,生物发光信号逐渐减弱.结论 Luc报告基因通过腺病毒载体成功转入BMSC,实现了光学报告基因成像对移植干细胞的示踪.

摘要： 10.3969/cmba.j.issn.1673-713X.2012.05.003%　　目的构建含有 p53蛋白结合位点的 p21或 survivin 基因启动子区的荧光素酶报告基因重组质粒，并获得稳定转染的细胞模型用于抗肿瘤化合物筛选.方法将含有 p53蛋白结合位点的 p21或 survivin 启动子区序列连接到 pGL4.17-basic 载体上，分别获得报告基因重组质粒 pGL4.17-p21和 pGL4.17-survivin；将两种重组质粒分别转染 A549细胞，经 G418筛选获得稳定转染细胞株 A549-p21和 A549-survivin；对该细胞模型进行条件优化，并将可调节 p53蛋白表达的多种抗肿瘤药物作用于稳定转染细胞，通过荧光素酶检测和 western blot 方法验证细胞模型用于药物筛选的可行性.结果成功建立了分别含有 p21或 survivin 启动子报告基因的 A549-p21和A549-survivin 稳定细胞模型，多种抗肿瘤药物作用该细胞后能够通过调节 p53蛋白表达而影响报告基因荧光素酶活性.结论构建的稳定转染细胞模型可以用于筛选以 p53为靶点的抗肿瘤化合物.

摘要： 本发明提供了一种含有肉牛DKK3基因3’UTR序列和双荧光素酶报告基因的质粒及其应用，所述质粒包含bta‑miR‑25与牛DKK3结合的靶点，其构建包括设计DKK3基因3’UTR区扩增引物并加入酶切位点Pme I和Xho I，以牛肾细胞(MDBK)cDNA为模板进行扩增，将PCR产物及pmirGLO Vector分别用限制性内切酶Pme I和Xho I进行双酶切，将酶切后纯化的载体pmirGLO Vector和DKK3基因3’UTR片段连接，转化，培养，PCR检测，测序，将测序正确的菌液重新扩大培养，质粒提取等步骤，本发明为筛选调控DKK3表达的miRNAs、评估miRNAs对DKK3表达的调控作用提供了简单易行的工具，具有灵敏度高、速度快和费用低等优点；本发明还为DKK3的表达调控带来了更深层次的认知，能运用于家畜肉质遗传改良分子调控机制的研究，具有较好的应用前景。

摘要： 本发明公开了一种基于基因编辑技术的监测miRNA表达的报告基因探针及构建方法。所述的探针包括pcDNA3.1载体、基因片段A和B，载体pcDNA‑CRE，载体pcDNA‑FLP。所述的基因片段A、B按照A‑B顺序与线性化的pcDNA3.1载体重组，所述的片段A由Loxp/FRT、Fluc、和5xmiR targets序列组成，所述的片段B为Loxp/FRT。本发明的报告基因分子影像探针基于Loxp/FRT‑CRE/FLP基因编辑技术结合报告基因分子显像技术，可以实时监测活体miRNA的表达变化，为活体miRNA表达的动态变化提供了一种实时、无创的监测工具。

摘要： 本发明提供了一种含WSB1基因启动子和报告基因的重组质粒及其构建方法和应用。具体地，克隆WSB1基因启动子特异性序列，重组到含萤火虫荧光素酶报告基因载体序列中，构建含WSB1基因启动子和报告基因的重组质粒，再将构建的重组质粒与海肾荧光素酶报告基因质粒pRL‑TK共转染肿瘤细胞，使其稳定表达，最后通过检测双荧光素酶的活性来反映WSB1基因启动子启动转录的活性，从而应用于靶向筛选抗肿瘤药物。本发明构建的重组质粒采用的是WSB1基因启动子，并选取了特定的启动子序列，所构建的药物筛选模型具有高特异性、高灵敏度和高准确度，实现了以WSB1为靶点的抗肿瘤药物的高通量筛选。

摘要： 本发明提供了一种人DNMT1基因3’‑UTR的荧光素酶报告基因载体及其构建方法和应用，该荧光素酶报告基因载体包括荧光素酶报告基因载体和DNMT1基因的3’‑UTR区核苷酸片段，荧光素酶报告基因载体为pMIR‑REPORT™，DNMT1基因的3’‑UTR区核苷酸片段的核苷酸序列如下所示：该荧光素酶报告基因载体的构建方法，其包括如下步骤：将经限制性核酸内切酶酶切的DNMT1基因的3’‑UTR区核苷酸片段，通过连接酶，连接到经相同的限制性核酸内切酶酶切的荧光素酶报告基因载体中，通过筛选获得人DNMT1基因的3’‑UTR荧光素酶报告基因载体。

摘要： 本发明提供了一种基于猪NLRP6基因启动子区的双荧光素酶报告基因载体，属于基因工程技术领域，将NLRP6基因启动子连接到pGL3‑Basic质粒中，得到双荧光素酶报告基因载体；所述NLRP6基因启动子的核苷酸序列如SEQ ID No.1或SEQ ID No.2所示。本发明明确了NLRP6基因启动子在NLRP6基因中的具体位置，将NLRP6基因启动子连接到pGL3‑Basic质粒中，得到双荧光素酶报告基因载体具有转录活性。

摘要： 本发明提供了一种用于监测gtfD和ftf基因水平表达上调的荧光素酶报告基因菌株、制备方法及其应用，涉及生物学技术领域。本发明所构建的用于监测gtfD和ftf基因水平表达上调的荧光素酶报告基因菌株能够通过检测荧光素的强弱来判断gtfD和ftf的表达情况，进而通过动态检测这两个基因表达可监测VicK磷酸酶的活性状态，以此来筛查阻断VicK磷酸酶活性的潜在小分子化合物或药物。本发明的荧光素酶报告基因菌株检测流程简单，技术敏感性低，同时能够大大节省检测时间和试剂成本。

摘要： 本发明公开用于体外筛选引起基因沉默药物的报告基因组、试剂盒及其应用，属于采用生物技术进行药物筛选技术领域。基于NGD过程，以及现有基因药物研发现状，将药物引起的NGD现象，构建一种报告基因组，其中，报告基因组包括：报告基因1：依次连接的泛素和报告基因A；报告基因2：依次连接的泛素、报告基因B、IRES和待降解蛋白编码区；报告基因A为荧光素酶基因、荧光蛋白基因或有色蛋白基因，报告基因B为荧光素酶基因、荧光蛋白基因或有色蛋白基因，报告基因A≠报告基因B。可实现体外筛选引起基因沉默的药物，最终达到治疗疾病的目的。

摘要： 本发明公开了一种含rfp报告基因的细菌基因敲除质粒及其应用，属于分子生物学领域。具体地，本发明提供一种质粒pBRR50，其含有庆大霉素抗性基因、mob接合转移元件、R6K复制起始位点、多克隆位点区和由点突变lac启动子控制的红色荧光蛋白编码基因rfp；所述lac启动子的碱基序列优选如SEQ ID NO.2所示。本发明还提供了上述质粒pBRR50的构建方法及应用，该质粒能高效表达红色荧光蛋白，使细菌在日光下呈现明显的红色。在基因敲除过程中，发生第一次同源重组的细菌具有庆大霉素抗性和红色菌落表型，发生第二次同源重组的细菌从红色恢复为原来的颜色，可根据菌落颜色对发生第二次同源重组的菌株实现直接的筛选。

摘要： 本发明公开了一种荧光报告基因元件、基因编辑监测系统及用途。所述基因元件包括依次连接的第一编码区、第二编码区和第三编码区；所述第一编码区包括第一荧光蛋白报告基因，所述第二编码区包括第二荧光蛋白报告基因，所述第三编码区包括第三荧光蛋白报告基因；所述第一荧光蛋白报告基因、第二荧光蛋白报告基因和第三荧光蛋白报告基因分别位于不同的阅读框中，以使仅一种荧光蛋白能正确表达。本发明的基因元件通过对目标DNA编辑结果进行实时监控，能确定最佳的编辑时间窗；通过对不同荧光发光细胞数量统计完成对目标DNA编辑结果的预测。此外，还能通过建立人源化细胞模型，进行疾病的基因治疗新技术的评估。

摘要： 一种Glb1重组报告基因，其包括在Glb1基因的终止密码子位点从5’至3’端方向顺序地插入的2A序列以及报告基因，所述Glb1重组报告基因能够将Glb1转录水平与报告基因的转录水平显著性关联。一种制备转基因报告小鼠的方法，所述方法包括：将构建体引入小鼠胚胎干细胞或者小鼠生殖细胞，获得包括Glb1重组报告基因的小鼠胚胎干细胞或者小鼠生殖细胞；并且利用所述包括Glb1重组报告基因的小鼠胚胎干细胞或者小鼠生殖细胞生成杂合子转基因报告小鼠。其中，所述Glb1重组报告基因具有由SEQ ID NO.1所示的核苷酸序列。