Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap／GFP Fusion Protein in Mammal Cells

s-Lap is a new gene sequence from pig retinal pigment epithelial (RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.     

ENHANCEMENT OF ULTRAVIOLET-DNA REPAIR IN den V GENE TRANSFECTANTS and T4 ENDONUCLEASE V-LIPOSOME RECIPIENTS

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and into wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells.     

分子信标用于p53 mRNA的体外定量检测

根据p53基因的序列设计并合成了能特异性检测p53 mRNA的分子信标(MB), 发展了一种快速定量测定细胞内总RNA提取物中p53 mRNA的方法. 采用鼻咽癌(CNE2)细胞系和经RNA干扰技术降低p53基因表达的CNE2-p53RNAi细胞系, 抽提总RNA并用MB检测, 验证了MB的检测对象是p53 mRNA. 将该方法应用于多种肿瘤细胞内p53基因表达水平的分析, 表达变化趋势与经典的mRNA分析方法RT-PCR检测结果相符. 在此基础上, 用MB对5-氟尿嘧啶(5-Fu)处理的肺腺癌细胞(A549)进行了p53 mRNA的体外定量检测, 结果表明采用MB能够快速地获知该药物对细胞内p53 mRNA表达影响的信息.     

INHIBITED NEOPLASTIC PHENOTYPE BY THE c-Ha-ras ANTISENSE RNA

A 2.0 kb fragment DNA plasmid which expresses antisense to the upstream first exon of c-Ha-ras oncogene was transfected into Ha-ras transformed cell lines, GCM-3T3 and REF-4.3. The transfection leads to the inhibition of malignant behaviour, shown by decreasing of growth speed, colony forming ability on soft agar, tumorigenicity in nude mice and increasing ot differentiation degree. In GCM-3T3 cells the lung metastasis frequency became much less (from 60% to 12.5%) after the transfection and the expression of ras oncogene product, and p21 protein was obviously decreased in the transfected cells. This work has first shown the inhibitory effect of antisense RNA on neoplastic behaviour in China.     

TNFa在血管内皮细胞ECV304中的靶向表达研究

在缺失了3'LTR U3区内病毒的启动子／增强子序列的逆转录病毒载体pLXSNd中,用血管内皮生长因子受体KDR的特异性启动子调控了TNFa在血管内细胞ECV304中的靶向表达。将构建的载体pLXSN-TNFa,pLXSNd-KDRp-TNFa和空载体pLXSN用PA317细胞包装后获得重组病毒,并用重组病毒分别感染NIH3T3细胞和ECV304细胞,培养物上清的ELISA结果证明,KDR启动子指导的TNFa在KDR阳性细胞ECV304中的表达量为在KDR阴性细胞NIH3T3中的表达量的8倍;而TR指导的TNFa在这两种细胞中的表达无明显差异,实现了TNFa在血管内皮细胞中的靶向表达,这可能为肿瘤基因治疗提供新途径。     

A novel sensing platform using aptamer and RNA polymerase-based amplification for detection of cancer cells

Cancer is one of the most serious and lethal diseases around the world. Its early detection has become a challenging goal. To address this challenge, we developed a novel sensing platform using aptamer and RNA polymerase-based amplification for the detection of cancer cells. The assay uses the aptamer as a capture probe to recognize and bind the tumor marker on the surface of the cancer cells, forming an aptamer-based sandwich structure for collection of the cells in the microplate wells, and uses SYBR Green II dye as a tracer to produce strong fluorescence signal. The tumor marker interacts first with the recognition probes which were composed of the aptamer and single-stranded T7 RNA polymerase promoter. Then, the recognition probe hybridized with template probes to form a double-stranded T7 RNA polymerase promoter. This dsDNA region is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the SYBR Green II dye and a standard fluorometer, resulting in the amplification of the fluorescence signal. Using MCF-7 breast cancer cell as the model cell, the present sensing platform showed a linear range from 5.0 × 10² to 5.0 × 10⁶ cells mL⁻¹ with a detection limit of 5.0 × 10² cells mL⁻¹. This work suggested a strategy to use RNA signal amplification combining aptamer recognition to develop a highly sensitive and selective method for cancer cells detection.     

An RNA polymerase I-driven human respiratory syncytial virus minigenome as a tool for quantifying virus titers and screening antiviral drug

Human respiratory syncytial virus(RSV) is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I(Pol I) was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence(GS), reverse complementary copy of Gaussia luciferase(Gluc) gene, gene end sequence(GE), and leader region in the direction of 5'–3'end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.     

Validation of phage T7 biological dosimeter by quantitative polymerase chain reaction using short and long segments of phage T7 DNA

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.     

纳米二氧化钛对卵巢肿瘤细胞的选择性凋亡的诱导作用

以人肾上皮细胞293T和中国仓鼠卵巢肿瘤细胞CHO为研究对象, 通过MTT法酶标仪检测、倒置显微镜观察和DNA ladder实验等方法, 研究了锐钛型TiO₂对它们的选择性凋亡诱导作用. 实验结果表明, 纳米TiO₂对肿瘤细胞具有非常明显的毒性作用, 可侵入细胞内部, 诱导CHO细胞产生细胞凋亡. 对于293T细胞, 虽然会抑制其增殖, 但不能够进入细胞膜内, 没有细胞凋亡现象的发生. 这一结论为利用纳米TiO₂治疗癌症提出了一条新思路.     

Synthesis of P-Chiral [ 18 O]-Labeled Isotopomeric Oligo(deoxyribonucleoside Phosphorothioate)s and Phosphates

Diastereomerically pure, isotopically labeled 5'- O -DMT-nucleoside-3'- O -(2-thio-4,4-"spiro"-pentamethylene-1,3,2-[ 18 O]oxathiaphospholane)s and -(2-oxo-4,4-"spiro"-pentamethylene-1,3,2-[ 18 O]oxathiaphospholane)s suitable for stereocontrolled synthesis of P-chiral oligonucleotide [ 18 O]phosphorothioates and [ 18 O]phosphates were synthesized. Obtained in that way "chimeric" oligonucleotide d[A PO A PS-Rp,Sp A PS-Rp , Sp A PS-Rp C PS(18O)-Rp G PS-Rp T PS-Rp T PS-Rp,Sp T PO T] was used for determination of the stereochemistry of hydrolysis by endonuclease from Serratia marcescens .     

Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system

The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system. The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction. The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus. The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization. Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained. Reusability of the immobilized mRNA as genetic information was also examined.     

Construction and Characterization of Lentiviral shRNA Expression Vector Targeting Rat CD40 Gene in Dendritic Cells

To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells(DCs), specific siRNA targets with short hairpin frame were designed and synthesized according to the mRNA sequence of rat CD40 gene. DNA oligo was cloned into lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. The verified plasmids were transfected into 293T cells that over-express recombinant CD40 in order to select the mos...     

Expression of Hepatitis B Surface Antigen Gene in Ginseng Cells

The recombinant plasmid pBIBSa containing the HBsAg DNA fragment was transferred into Agrobacte-rium tumefaciens strain LBA4404 directly. Ginseng cells were transfected with A. tumefaciens carrying pBIBSa and the ginseng cell lines carrying HBsAg-S gene were obtained. The presence of target gene in the transfect cells was confirmed by PCR and RT-PCR. A clear band at the site of 700 bp was observed by agarose electrophoresis analysis of the samples containing the target gene. HBsAg expressed by the transgeni...     

侧链含咪唑取代基聚(L-天冬酰胺)高效基因载体研究

通过氨基引发聚丁二酰亚胺( PSI)开环反应,制备了系列侧链含氨乙基和咪唑丙基的聚(L-天冬酰胺)共聚物(P1 ～ P5).该系列聚合物不仅具有极低的细胞毒性,而且随侧链中咪唑取代基含量的增加,聚合物在pH 5 ～8范围内缓冲能力显著提高.通过凝胶电泳、粒径和电位分析等研究了聚合物与质粒DNA的相互作用.结果表明,所有...     

Toehold-Mediated Strand Displacement in a Triplex Forming Nucleic Acid Clamp for Reversible Regulation of Polymerase Activity and Protein Expression

Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleic acids, target affinity and biological stability superior to that of natural DNA may be obtained. This work demonstrates how a chimeric clamp consisting of acyclic (L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formation of a highly stable triplex structure. The (L)-aTNA clamp is released from the target again by the addition of a releasing strand in a strand displacement type of reaction. It is shown that the clamp efficiently inhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully applied to the regulation of luciferase expression by reversible binding to the mRNA. When targeting a sequence in the double stranded plasmid, 40 % downregulation of protein expression is achieved.     

HEMATOPORPHYRIN DERIVATIVE-INDUCED PHOTODYNAMIC INHIBITION OF Na+/K+-ATPase IN L929 FIBROBLASTS, CHINESE HAMSTER OVARY CELLS AND T24 HUMAN BLADDER TRANSITIONAL CARCINOMA CELLS

The photodynamically induced inhibition of Na⁺/K⁺-ATPase with hematoporphyrin derivative as photosensitizer was studied in murine L929 fibroblasts, Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells. In T24 cells the inhibition of the enzyme activity appeared to be caused by ATP depletion rather than by direct damage from the enzyme itself. In L929 and CHO cells, on the other hand, the inhibition was caused by photodynamic damage from the enzyme molecule. For all three cell lines it was shown that a causal relationship between photodynamically induced reduction in Na⁺/K⁺-ATPase activity and the loss of clonogenicity is highly unlikely.