The Anopheles gambiae alpha-tubulin-1b promoter directs neuronal, testes and developing imaginal tissue specific expression and is a sensitive enhancer detector

A knowledge gap in mosquito functional genetic analysis is the dearth of characterized regulatory regions that can target tissue specific transgene expression. To broaden the tools available, a promoter region of the Anopheles gambiaeα-tubulin1b gene has been assayed following fusion to the green fluorescent protein (GFP) reporter gene and stable transformation of An. gambiae. In eight transgenic lines, the Angtub α1b regulatory region directed a core profile of tissue specific expression in the head, chordotonal organs, ventral nerve cord and testes. This profile overlaps those seen for α2-tubulin expression in Drosophila melanogaster and Bombyx mori. In addition, widespread position dependant expression was observed in other specific tissues that were unique to each line. For example, in different lines, expression was observed in larval and adult muscles, fatbody, cuticle and midgut secretory cells. The majority of genomic transgene insertions were mapped to within 10 kb of a gene, suggesting that the Angtub α1b basal promoter is particularly sensitive to enhancers and may be suitable to form the basis of a sensitive enhancer trapping construct, in combination with a binary expression system such as Gal4-UAS.     

用基于galK的同源重组的方法构建arc基因表达增强的载体

目的:利用同源重组的方法,将arcBAC(bacteria artificial chromosome)启动子区域的SRE(serumre-sponse element)改造成Gal4结合序列,以此通过Gal4/UAS系统增强arc基因表达。方法:在大肠杆菌中利用依赖于噬菌体基因的同源重组,通过galK正负筛选的方法改造arcBAC。结果:成功将arc启动子区域的SRE改造成Gal4结合序列,并且没有影响arc基因的其他序列。结论:成功改造BAC,可以将此BAC用在Gal4/UAS系统增强arc基因表达的实验中。     

调控组织因子基因表达的药物筛选研究

本研究建立TF启动子转录活性的荧光素酶基因稳定细胞株,应用该细胞模型筛选调控TF基因表达的药物,并为深入研究其分子机制打下基础。构建TF启动子的一系列5′端截短型的荧光素酶报告基因质粒(包括-2174 bp~+128 bp,-684 bp~+128 bp,-247 bp~+128 bp,-201 bp~+128 bp),将质粒电转染至U937细胞中,建立表达荧光素酶报告基因的稳定细胞株。应用ATRA验证该细胞株的功能;应用bortezomib、尿多酸肽(CDA-II)等药物处理该细胞株24小时,分析荧光素酶基因活性,筛选出能够调控TF基因表达的药物。结果发现,5 nmol/L bortezomib能激活其转录活性,上调TF转录本表达水平;1 mg/ml CDA-Ⅱ抑制TF启动子的转录活性,下调TF转录本的表达水平。TF启动子逐步截短功能分析发现,bortezomib及CDA-ⅡII调控TF启动子转录活性的区域位于-201 bp—0 bp之间。结论:本研究建立了表达TF启动子荧光素酶活性的U937稳定细胞株,并筛选出能够调控TF基因转录的药物CDA-II及bortezomib,为将来筛选新药物及深入研究其分子机...     

Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer.

Minicircles are a new form of supercoiled DNA molecule for nonviral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy. They were obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. We produced two minicircles containing the luciferase or beta-galactosidase gene under the control of the strong human cytomegalovirus immediate-early enhancer/promoter. Comparing maximal differences, these minicircles gave 2.5 to 5.5 times more reporter gene activity than the unrecombined plasmid in the NIH3T3 cell line and rabbit smooth muscle cells. Moreover, injection in vivo into mouse cranial tibial muscle, or human head and neck carcinoma grafted in nude mice resulted in 13 to 50 times more reporter gene expression with minicircles than with the unrecombined plasmid or larger plasmids. Histological analysis in muscle showed there were more transfected myofibers with minicircles than with unrecombined plasmid.     

A cytoskeletal actin gene in the mosquito Anopheles gambiae

Five actin genes have been identified in the mosquito Anopheles gambiae , and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5 and 3 flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeietal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeietal actin genes of Droso-phila melanogaster , and in sequence, equally similar to the Artemia cytoskeietal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated actWior its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.     

Glioma-specific and cell cycle-regulated herpes simplex virus type 1 amplicon viral vector

We have engineered a novel herpes simplex virus type 1 (HSV-1)-based amplicon viral vector, whereby gene expression is controlled by cell cycle events. In nondividing cells, trans-activation of the cyclin A promoter via interaction of the Gal4/NF-YA fusion protein with the Gal4-binding sites is prevented by the presence of a repressor protein, cell cycle-dependent factor 1 (CDF-1). CDF-1 is specifically expressed during the G(0)/G(1) phase of the cell cycle and its binding site is located within the cyclin A promoter. In actively proliferating cells, trans-activation could take place because of the absence of CDF-1. Our results showed that when all these cell cycle-specific regulatory elements are incorporated in cis into a single HSV-1 amplicon plasmid vector backbone (pC8-36), reporter luciferase activity is greatly enhanced. Transgene expression mediated by this series of HSV-1 amplicon plasmid vectors and amplicon viral vectors could be regulated in a cell cycle-dependent manner in a variety of cell lines. In a further attempt to target transgene expression to a selected group of actively proliferating cells such as glial cells, we have replaced the cytomegalovirus promoter of the pC8-36 amplicon plasmid with the glial cell-specific GFAP enhancer element. With this latter viral construct, cell type-specific and cell cycle-dependent transgene expression could subsequently be demonstrated specifically in glioma-bearing animals. Taken together, our results suggest that this series of cell cycle-regulatable HSV-1 amplicon viral vectors could potentially be adapted as useful tools for the treatment of human cancers.     

Facilitating fructose-driven metabolism exerts a protective effect on anoxic stress in Drosophila

Hypoxic stress is linked to various cardiovascular disorders (e.g., stroke, myocardial infarction), mediated, at least in part, by a reduction in ATP synthesis. Fructose-driven glycolysis is proposed as an alternative pathway capable of sustaining ATP production even under anoxic conditions. Here, we tested the hypothesis that facilitating fructose-driven metabolism exerts a protective effect against anoxic stress in Drosophila. Genetically modified flies with the human fructose transporter (GluT5) and ketohexokinase (KHK) genes downstream of upstream activating sequence (UAS) were constructed. The GAL4-UAS system was confirmed to: (i) increase the expression of GluT5 and KHK in a tissue-specific and a time-dependent manner (i.e., whole flies [with Act5c-gene switch GAL4 driver], neurons [with elav-gene switch GAL4 driver]) and (ii) reduce mortality of flies when placed under anoxic stress. Taken together, these data suggest that increasing fructose metabolism may be a clinically relevant approach to minimize hypoxia-induced cellular damage.     

人端粒酶反转录酶重组绿色荧光表达载体转染椎间盘正常髓核细胞

背景：椎间盘髓核细胞分离培养困难,老化较快,迫切需要一种标准细胞株用于实验研究。目的：探讨人端粒酶反转录酶重组绿色荧光表达载体的构建及其转染正常髓核细胞构建永生化细胞的可行性研究。方法：通过目的基因克隆、真核表达质粒中目的基因序列测定、目的基因真核表达质粒的构建、转染人端粒酶反转录酶表达检测等步骤进行实验。结果与结论：构建出人端粒酶反转录酶重组绿色荧光表达载体,成功转染正常髓核细胞并在细胞中稳定表达。结果表明运用人端粒酶反转录酶转染椎间盘髓核细胞构建永生化细胞是一种可行的方法。     

MTS1基因β启动子转录活性研究

目的 研究IMTSl基因β启动子在急性T淋巴细胞白血病细胞株中的转录激活情况，鉴定β启动子转录活性的功能片段区。方法 用DNA重组方法，构建MTS1基因口启动子3′端转录起始点相同，而5′端序列不同的7种pGL3重组质粒。用脂质体介导的基因瞬时转染法，将构建的重组质粒分别转染MTSl基因双等位缺失的Jurkat细胞株，检测pGL3重组质粒中荧光素酶报告基因的表达，观察β启动子在Jurkat细胞中的激活情况及其基础转录活性片段区。结果 成功构建了MTSI基因β启动子7种不同片段的重组质粒，它们在Jurkat细胞中均有转录活性，其中0．38kb Sac Ⅱ-Sac Ⅰ酶切片段是MTSl基因β启动子转录活性的基础片段。结论 IMTSlβ启动子可在Jurkat细胞中被激活。     

含CXCL12基因的重组腺病毒载体的构建及鉴定

目的:构建并鉴定含小鼠CXCL12基因和EGFP基因的重组腺病毒载体,并观察其表达。方法:利用腺病毒载体系统Adeno-X,通过质粒抽提、电泳、酶切、连接、转化等基因工程技术,构建重组腺病毒质粒pAdeno-CXCL12-EGFP;酶切、PCR鉴定;利用293细胞包装pAdeno-CXCL12-EGFP,荧光倒置显微镜观察EGFP的表达。结果:成功构建重组腺病毒质粒pAdeno-CXCL12-EGFP,转染pAdeno-CXCL12-EGFP的293细胞内可见EGFP表达。结论:利用腺病毒载体系统Adeno-X成功构建含CMV启动子的CXCL12和EGFP基因的腺病毒载体。