CKLFSF1基因与CKLFSF2基因间存在的顺式作用元件

探讨趋化素样因子超家族成员 1,2基因 (CKLFSF1基因与CKLFSF2基因 )间的短序列对其下游基因表达的调控作用 .运用PCR技术扩增CKLFSF1基因与CKLFSF2基因间的序列 ,将此片段插入含有萤光素酶 (luciferase)报告基因载体上 .以磷酸钙介导基因转染技术 ,将重组质粒以及阴性和阳性对照组质粒转染到HeLa细胞 ,进行瞬时表达分析 .在pGL3 Basic质粒中的报告基因萤光素酶无表达 ,但将CKLFSF1与CKLFSF2基因间的序列插入到启动子上游或下游后 ,显著抑制其下游基因的表达 ,萤光素酶活性明显降低 .结果提示 ,CKLFSF1与CKLFSF2基因间的序列不具有启动子活性 ,但是该序列对其下游基因表达具有负调控作用     

表达萤光素酶的HIV药物筛选细胞模型的建立

目的:构建基于萤光素酶的单次复制人免疫缺陷病毒(HIV)细胞模型,用于抗HIV药物的筛选。方法:构建含萤光素酶报告基因的假型慢病毒质粒,将疱疹性口炎病毒外膜糖蛋白(VSV-G)的表达质粒、HIV-1 Rev蛋白表达质粒、HIV Gag-Pol蛋白表达质粒和含萤光素酶报告基因的重组慢病毒质粒共转染HEK 293FT细胞,制备假型慢病毒;在假型慢病毒生产和再感染新鲜HEK 293FT细胞的过程中加入逆转录酶和蛋白酶抑制剂(如AZT),检测再感染的细胞中萤光素酶的表达水平,从而判断药物对HIV的抑制作用。结果:构建了含萤光素酶报告基因的重组慢病毒质粒pLenti-Luc;利用已知抗HIV药物AZT进行测试,发现HIV药物处理组细胞中萤光素酶活性远低于对照组。结论:建立了基于萤光素酶的HIV药物筛选细胞模型,该系统使用单次复制的报告病毒,具有良好的安全性,而使用萤光素酶基因作为报告基因使该系统具备极高的敏感性,该系统适合于进行高通量药物筛选。     

含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体的制备与表达分析

目的:制备含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体,为慢病毒载体的进一步广泛应用奠定基础。方法:克隆构建含分泌型萤光素酶和绿色荧光蛋白双报告基因的转基因载体pCS-gluc-2A-eGFP,酶切与序列分析鉴定其正确后,与包装质粒pCMVHR’Δ8.2、包膜质粒pVSV-G共转染293FT细胞,获得含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体;重组慢病毒载体感染A549、Huh7细胞后,用荧光显微镜直接观察报告基因GFP的表达,或取细胞上清实时检测分泌型萤光素酶的表达。结果:制备了含双报告基因的重组慢病毒载体,感染细胞后可以活体观察绿色荧光蛋白的表达,也可以快速灵敏地检测到分泌型萤光素酶的表达。结论:所获含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体感染效率高,表达易于活体实时检测,灵敏度高。本研究为慢病毒载体的广泛应用奠定了基础。     

人免疫缺陷病毒假病毒药物筛选模型的构建及其应用

目的:构建人免疫缺陷病毒(HIV)假病毒模型,用多种HIV逆转录酶和蛋白酶抑制剂作用于该模型,以检测其是否能有效用于HIV抑制药物的筛选。方法:通过载体改造获得最终慢病毒载体puc18-NL4-3-LUC-stop,其中含有萤光素酶基因,将该载体与包膜质粒VSV-G共转染293FT细胞,包装产生HIV假病毒,在假病毒包装和病毒感染293FT细胞的过程中加入蛋白酶和逆转录酶抑制剂,通过检测感染细胞中萤光素酶的表达来检测该模型的有效性,并利用此模型检测药物的抗病毒效果。结果:将HIV逆转录酶和蛋白酶抑制剂作用于该假病毒模型时发现萤光素酶的表达得到很大程度的抑制。结论:建立了HIV假病毒药物筛选模型,该模型以萤光素酶基因作为报告基因,快速灵敏,在抗HIV药物筛选中有一定的应用价值。     

miR-126报告基因载体的构建及其功能鉴定

目的:根据miR-126的预测靶点构建荧光素酶报告基因重组质粒,并进行功能鉴定。方法:利用sanger数据库提供的miR-126靶序列设计引物,PCR扩增目的微小RNA(microRNAs,miRNAs)靶基因3'非编码区(three-prime untranslated regions,3'UTRs)序列,PCR产物双酶切,后连入经过同样双酶切的pGL3-control载体中,连接产物转化大肠杆菌DH5α,进行阳性克隆鉴定。同样,将候选靶基因3'UTRs突变,突变型3'UTR克隆入pGL3-control报告载体,构建野生型和突变型的报告基因重组质粒。将野生型和突变型的报告基因载体分别和化学合成的microRNA以及内参质粒共转染293TN细胞,进行双荧光素酶检测。结果:成功构建miR-126报告基因野生型和突变型重组质粒pGL3-VEGF-A-3'UTR和pGL3-VEGF-A-3'UTR,质粒测序及酶切结果完全正确。瞬时转染实验显示,过表达miR-126能直接抑制VEGF-A-3'UTRs报告基因活性。结论:miR-126对VEGF-A具有靶向调节功能。     

猪防御素PD基因表达载体的构建

目的：构建猪防御素PD基因表达载体。方法：化学合成经过适当改造的带有双酶切位点的PD基因,将该基因定向插入带有相同酶切位点的融合表达质粒PinPoin^TMXa-3的多克隆位点构建表达质粒。结果：构建的表达载体经双酶切电泳分析及插入基因片段序列分析,表明PD基因表达载体构建成功。结论：PD基因表达载体的构建,为进一步获得PD基因的表达产物,研究其抗菌活性、抗菌机理打下基础。     

MDR1基因单靶点双荧光素酶报告基因系统的建立

目的通过构建以MDR1启动子为启动序列的双荧光素酶报告基因系统并进行活性分析,为MDR1基因表达的单靶点调控研究和逆转剂的筛选提供一种有效的方法。方法从HCT-8细胞中提取DNA并克隆含有MDR1基因启动子中Y—box序列。将该序列重组到萤光素酶报告基因载体pGL-3．Basic的启动区域中,从而构建报告基因载体pGL-MDR1。将pGL-MDR1和pRL-TK载体共转染到HCT-8和HCT-8／VCR细胞中。通过调节不同载体的比例来优化转染效率。利用MDRI基因激活剂（热诱导）和抑制剂（EGCG）等处理来分析其启动转录活性受外界因素的影响。结果通过直接测序法验证了pGL-MDR1含有MDR1基因启动子Y—box序列且没有出现碱基突变。在pGL-MDR1和pRL-TK的转染比例为5：5时,转染效率最高并具有最高的萤光素酶活性。通过MDR1基因激活处理后表现为时间依赖性地激活MDR1基因的表达,而MDR1基因抑制剂的作用则相反。结论MDR1启动子为启动序列的双荧光素酶报告基因系统建立成功。该系统不但可以用于研究活体生物发光成像和MDRI基因表达的机理,而且可用于单靶点的多药耐药抑制剂的筛选。     

靶向Smad4基因shRNA质粒表达载体的构建及筛选

目的:构建编码Smad4mRNA的shRNA真核表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体.方法:根据GenBank提供的人Smad4基因的mRNA序列构建2个shRNA质粒表达栽体和1个阴性对照质粒表达载体,并通过基因测序进行鉴定.经鉴定后转染体外培养人成纤维细胞,western-blot检测Smad4蛋白水平抑制表达效果.结果:构建质粒表达载体测序结果显示,插入片段测序结果与合成的shRNA序列一致;靶向Smad4基因shRNA质粒表达栽体对所转染的人成纤维细胞中Smad4蛋白水平表达均有抑制作用,其中shRNA1最为明显.结论:成功构建了靶向Smad4基因shRNA质粒表达栽体,其中抑制Smad4基因表达效果最为明显的是shRNA1,为进一步研究Srnad4基因的RNA干扰奠定了基础.     

人p53启动子萤光素酶报告基因的构建及其活性测定

目的：克隆p53基因的启动子,插入萤光素酶报告基因载体,并检测启动子活性。方法：采用PCR技术从人肝癌细胞系HepG2基因组中扩增人p53启动子,插入萤光素酶报告基因载体pGL4．0-empty,将重组质粒转染293T、ZR75-1、HepG2、A549细胞,测定p53启动子的转录活性。结果：构建了p53启动子的萤光素酶报告基因;通过测序及质粒酶切鉴定,所构建的p53启动子正确;活性实验表明,报告基因在多种细胞中显示构建的p53启动子活性,并呈现一定的剂量效应;转录因子USF能以剂量效应方式提高p53报告基因的转录活性。结论：克隆了人p53启动子,为进一步研究调控p53的转录因子奠定了基础。     

抑制Dicer基因对shRNA功能发挥的影响

本文将Dicer基因的RNA酶III结构域作为靶区,设计并构建了两个抗Dicer基因的小发夹样RNA(shRNA)表达载体,将其转染2215、结肠癌TC细胞和基因组中整合有绿色荧光蛋白基因(GFP)的HepG2A9细胞,通过RT-PCR评价RNA干扰抑制Dicer基因表达的效率;当HepG2A9细胞Dicer基因表达被上述RNA干扰抑制时,再转染抗GFP的shRNA表达载体,通过RT-PCR和荧光显微镜观察GFP表达水平。结果显示,在不同细胞系中,这两个抗Dicer基因shRNA表达载体,均能明显抑制Dicer基因的表达;当Dicer基因受抑时,后续转染抗GFP的shRNA表达载体不能有效抑制GFP的表达。结果表明,抗Dicer基因shRNA表达载体,能够明显抑制Dicer基因的表达;shRNA表达载体的功能发挥需要Dicer酶的直接参与。     

An advanced blue-white screening method for construction of shRNA expression vectors

Short hairpin RNA (shRNA) encoded within an expression vector is an effective tool for exploration of gene function in mammalian cells. Many of the current methods for constructing shRNA expression vectors require cumbersome and time-consuming procedures for identification of the desired recombinants. We have developed a highly efficient and less labor-intensive cloning method that allows the construction of shRNA expression vectors in one day and with minimal effort. This advanced blue-white screening technique was developed by combining the reconstitution of ideal lacO with TA cloning. The DNAs are simply ligated into the destination vectors and, following transformation, a desired recombinant event will give a typical blue colony. In addition, we have used this cloning method for the construction of targeting reporter expression vectors to measure the efficacy of the corresponding shRNA. We constructed 122 functional shRNA expression vectors and sequencing of the positive cloning vectors confirmed a high degree of accuracy. Only three short DNA primers are needed for constructing both shRNA and targeting reporter expression vectors. This advanced blue-white screening system is a powerful tool for the high-throughput assay of RNAi libraries.     

Optimization of a genome-wide disordered lentivector-based short hairpin RNA library

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro ^R, copGFP, or H-2K ^k as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.     

Stringent testing identifies highly potent and escape‐proof anti‐HIV short hairpin RNAs

Background

RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence‐specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV‐1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co‐transfection assay in which shRNA constructs were transfected with an HIV‐1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV‐1 sequences.

Methods

In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV‐1 molecular clone and also infected shRNA‐expressing T cell lines with HIV‐1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA‐expressing T cells upon challenge with increasing dosages of HIV‐1, and measured the dose required to result in massive virus‐induced syncytia formation in this 2‐week assay.

Results

Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co‐transfection assays. The use of increased dosages of HIV‐1 selected the same highly potent shRNAs as the laborious and extended escape study.

Conclusions

These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs. Copyright © 2009 John Wiley & Sons, Ltd.     

A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

hERG shRNA 表达载体构建及其稳定转染骨肉瘤细胞系MG-63、 SOSP-9607 的建立

目的:构建hERG钾离子通道蛋白(human ether-a-go-go-related gene potassium channel)shRNA表达载体质粒,获得稳定转染干扰质粒的人骨肉瘤细胞系MG-63、SOSP-9607。方法:将4对合成的寡核苷酸链退火形成双链,连接入pGPU6/GFP/Neo表达载体,并测序鉴定。使用脂质体法将重组的质粒转染至MG-63、SOSP-9607,通过G418筛选建立稳定转染的两种细胞系,采用免疫印迹(Western blot)技术检测hERG蛋白的表达。结果:测序结果证实shRNA与载体连接正确,免疫印迹实验证实hERG蛋白表达显著降低。结论:成功构建了hERG shRNA真核表达载体,获得了稳定表达hERG shRNA的人骨肉瘤细胞系MG-63和SOSP-9607。     

Silencing of mammalian genes by tetracycline-inducible shRNA expression

Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.     

In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene

ABSTRACT: BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined. RESULTS: Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency. CONCLUSIONS: Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.     

IRDL Cloning: A One-Tube,Zero-Background,Easy-to-Use,Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

Rapid and efficient construction of expression vectors and subsequent transformation are basic recombinant methods for the investigation of gene functionality. Although novel cloning methods have recently been developed, many laboratories worldwide continue to use traditional restriction digestion-ligation methods to construct expression vectors owing to financial constraints and the unavailability of appropriate vectors. We describe an improved restriction digestion-ligation (IRDL) cloning method that combines the advantage of directional cloning from double digestion-ligation with that of a low background observed by using a positive selection marker gene ccdB to facilitate digestion and ligation in a single tube. The IRDL cloning overcomes the time-consuming and laborious limits of traditional methods, thereby providing an easy-to-use, low-cost, and one-step strategy for directional cloning of target DNA fragments into an expression vector. As a proof-of-concept example, we developed two yeast vectors to demonstrate the feasibility and the flexibility of the IRDL cloning method. This method would provide an effective and easy-to-use system for gene cloning and functional genomics studies.     

猪Nanog基因四环素诱导干扰载体的构建和鉴定

Nanog基因是在早期胚胎和干细胞等多能性细胞中特异表达的重要基因,但有关猪Nanog基因功能的相关研究甚少。四环素诱导干扰载体是一种可通过四环素等药物条件性诱导干扰目的基因的载体,尤其适用于在发育过程中起着关键作用的基因沉默。常规的四环素干扰系统为二元载体,与一元载体相比获得针对特定基因干扰的稳定细胞系所需周期更长。首先通过构建pGenesil 1.0-shRNA重组干扰载体,瞬时转染稳定过表达猪Nanog基因的猪胎儿成纤维细胞后通过Realtime-PCR筛选出干扰效率可达80%以上的干扰片段。之后将筛选得到的干扰片段插入到改造的一元四环素诱导干扰载体TREsilencer,对稳定表达猪Nanog基因的猪胎儿成纤维细胞进行了瞬时转染。实验分别通过光密度检测以及Realtime-PCR检测了不同浓度doxycycline的诱导效率和干扰效率。结果表明,所构建的四环素诱导干扰载体TREsilencer-shRNA5随着四环素浓度的增加,诱导Nanog基因的干扰效率增加,在处理浓度为1μg/ml时干扰效率可达70%以上,为后续得到可诱导的稳定干扰猪Nanog基因的细胞系和进一步研究猪Nanog基因功能奠定了基础。