水稻瘤矮病毒S3和S8基因共表达杆状病毒转移载体构建及重组病毒的鉴定

为构建携带水稻瘤矮病毒(Rice gall dwarf virus,RGDV)主要内层衣壳蛋白S3基因和外层衣壳蛋白S8基因的重组杆状病毒,将目的基因(S3和S8)分别亚克隆到杆状病毒转移载体pFastBacDual多角体启动子(PH)和p10启动子的下游.经酶切和确证性序列测定,将其转化到DH10Bac感受态细胞中,获得重组杆粒rbpFBDS3-S8,采用脂质体转染法,将rbpFBDS3-S8转染草地贪夜蛾(Spodoptera frugiperda)Sf9细胞包装病毒,PCR筛选鉴定重组病毒.结果表明:Sf9昆虫细胞被侵染72 h后,倒置显微镜下观察到细胞增大,培养液和细胞内出现颗粒状物质,部分细胞破裂甚至裂解,说明S3和S8基因已整合到重组杆状病毒基因组中,这为开展RGDV主要结构蛋白在昆虫细胞中的表达及其功能研究奠定了基础.     

RNA干扰PTEN基因对HCT-8细胞增殖能力的影响

目的:观察小干扰RNA(siRNA)对结肠癌相关基因PTEN表达的抑制作用及其对结肠癌细胞增殖能力的影响.方法:构建携带有针对PTEN基因序列的siRNA真核绿色荧光表达载体重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4,脂质体法转染入结肠癌HCT-8细胞(分别为p1、p2、p3和p4组),同时设转染阴性对照质粒组(p5组)和亲本细胞组(p6组),实时 PCR方法检测PTEN mRNA表达,MTT法分析细胞增殖活性变化.结果:通过荧光显微镜观察计数,细胞转染率为58%.实时 PCR方法检测结果显示,siRNA 重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4的抑制率分别为48.3%、76.5%、29.4%和61.2%,与p6组比较差异均有统计学意义(P<0.05).MTT结果表明,细胞增殖抑制能力明显增强.结论:PTEN基因在肿瘤的发生发展中可能起到一定的作用.     

SCIRR10截短体真核表达载体的构建及其在COS-7细胞中的表达

目的:构建SCIRR 10蛋白3个截短体的哺乳动物细胞表达系统,探讨SCIRR 10蛋白的功能位点.方法:PCR扩增出SCIRR 10蛋白的3个功能截短体表达DNA序列,构建3种真核表达质粒载体pcDNA3.1/Myc-His-SCIRR 10(1-145aa)、pcDNA3.1/Myc-His-SCIRR 10(1-90aa)和pcDNA3.1/Myc-His-SCIRR 10(1-70aa).将3个表达载体质粒转染到COS-7细胞中,应用Western blotting 方法检测3个蛋白截短体的表达情况.结果:3个被截短的SCIRR 10蛋白相对分子质量分别为18 800、12 800和10 700.在COS-7细胞中可以成功表达3个SCIRR 10蛋白截短体.相对3个SCIRR 10截短体蛋白的表达量,β-tubulin蛋白无明显变化.结论:成功构建SCIRR 10蛋白3个截短体的哺乳动物细胞表达系统,通过此系统获得有生物活性的3个SCIRR 10截短体蛋白.     

特异性下调GRP78基因表达对卵巢癌细胞耐药性的影响

目的:探讨RNA干扰技术沉默葡萄糖调节蛋白78(GRP78)基因对人卵巢癌细胞耐药性的影响,阐明GRP78基因沉默逆转肿瘤细胞耐药性的生物学机制.方法:构建pSilencerTM3.0-H1-GRP78 siRNA重组质粒,脂质体介导转染至SKOV3/DDP细胞;RT-PCR和Western blotting法检测GRP78基因的蛋白的表达;Western blotting法检测caspase-4和caspase-3蛋白表达;流式细胞术检测细胞凋亡率.结果:转染GRP78 siRNA重组质粒的SKOV3/DDP细胞GRP78基因和蛋白表达较转染空质粒组明显降低(P<0.05);加用顺铂后,转染GRP78 siRNA重组质粒细胞组较未转染GRP78 siRNA重组质粒细胞组caspase-4和caspase-3表达明显增加(P<0.05),细胞凋亡率也明显增加(P<0.05).结论:抑制GRP78基因表达能通过上调caspase-4和caspase-3表达及增加顺铂诱导的SKOV3/DDP细胞凋亡率,降低SKOV3/DDP细胞对顺铂的耐药性.     

水稻硫转运蛋白基因OsST的亚细胞定位

[目的]构建水稻硫酸根转运基因OsST与YFP黄色荧光蛋白融合表达载体,对OsST蛋白进行亚细胞定位.[方法]从水稻叶片的cDNA中克隆OsST基因ORF全长,测序验证后连入pA7-YFP表达载体,通过基因枪将融合载体转入洋葱上表皮细胞,在激光共聚焦显微镜下观察细胞中荧光发光部位.[结果]OSST蛋白定位于细胞膜和核膜上.[结论]为进一步研究硫转运蛋白的功能及硫酸根运输的机理奠定了基础.     

鸡Mx蛋白在293T细胞中的表达及其抗病毒活性

[目的]研究鸡Mx 蛋白在293T 细胞中的表达并检测其抗病毒活性.[方法]将鸡的Mx基因克隆入pEGFP-C1栽体,经筛选鉴定后磷酸钙法转染293T 细胞,并对转染细胞进行荧光分析以及RT-PCR与Western blot鉴定,同时提取细胞蛋白质,微量细胞病变试验检测其抗新城疫病毒的活性.[结果]试验构建的pEGFP-C1-cMx真核表达栽体转染293T 细胞后,在胞浆中可检测到绿色荧光,RT-PCR与Western blot 结果进一步证实,pEGFP-C1-cMx可在293T细胞中表达,微量细胞病变试验显示,表达的融合蛋白可保护鸡胚成纤维细胞免受新城疫病毒感染.[结论]试验构建的pEGFP-C1-cMx 表达载体可在293T细胞中表达具有抗新城疫病毒活性的鸡Mx 蛋白.     

重组腺病毒介导血小板衍生生长因子感染人类脐带间质干细胞的实验研究

目的 检测重组腺病毒方法介导血小板衍生生长因子(PDGF)对人类脐带间质干细胞(MSC)的感染能力,为将其用于基因治疗奠定基础.方法 反转录-聚合酶链反应(RT-PCR)法扩增人类PDGF的cDNA序列,构建重组病毒载体AdPDGF.分离培养人类脐带MSC.体外以不同感染复数感染MSC后,流式细胞术检测感染效率,荧光显微镜观察绿色荧光蛋白(GFP)表达.锥虫蓝染色及四甲基偶氮唑蓝(MTT)法检测感染后细胞生存活力及增殖能力.酶联免疫吸附(ELISA)法检测细胞上清液中PDGF分泌水平.结果 成功构建病毒载体AdPDGF.其对MSC的感染效率随感染复数增加而增高,当感染复数为50时,达到最高,为87.36%.未感染的MSC、感染AdPDGF和对照病毒的MSC活力分别为(97.8 ±2.3)%、(91.9 ±4.0)%和(92.8 ±4.0)%.增殖能力分别为(100 ±16.8)%,(95.9±12.0)%和(87.5±9.7)%,感染AdPDGF的MSC与两个对照比较差异均无统计学意义(P>0.05).感染48 h后MSC能有效分泌PDGF,感染复数为10、30、50时,PDGF分泌水平分别为(1.53±0.37)、(3.03 ±0.68)和(5.25±0.92)ng/ml,MSC-GFP及MSC组未检测到有PDGF分泌.结论 成功构建AdPDGF重组腺病毒并高效感染人类脐带MSC.     

rAAV/CEA转染树突状细胞诱导特异性CTL杀伤MCF-7细胞系CD44+ CD24-/low乳腺癌干细胞

目的:携带癌胚抗原(carcinocmbryonic antigen,CEA)基因的重组人腺相关病毒(reconstructive human adeno-association virus,rh-AAV)感染树突状细胞(dendritic cell,DC)诱导获得抗原特异性细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL),体外检测其对CD44+CD24-/low乳腺癌干细胞的杀伤效果.方法:分离供者外周血单个核细胞,以细胞因子白介素-4(interleukin-4,IL-4)、粒细胞-巨噬细胞克隆刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)诱导培养获得DC,细胞因子白介素-2(interleukin-2,IL-2),刺激培养获得T淋巴细胞.含CEA基因的rh-AAV感染培养DC,诱导成熟后将DC和T细胞混合培养获得CTL细胞.流式分选MCF-7和MA-MDB-231细胞系中CD44+CD24-/low乳腺癌干细胞,MTT法检测CTL细胞对CD44+CD24-/low乳腺癌干细胞的杀伤效果.结果:MCF-7和MDA-MB-231中CD44+/CD24-/low群比例分别为5.1%和76.3%.CEA基因转染DC诱导的CTL细胞对表达CEA抗原的MCF-7杀伤率为46.5%±15.0%,与未转染组相比,差异有统计学意义(P=0.009);对MCF-7细胞中分选的CD44+CD24-/low乳腺癌干细胞的杀伤率为44.7%±28.2%,明显高于未转染组.对非乳腺癌干细胞杀伤率为50.6%±22.2%,与未转染组相比,差异有统计学意义(P=0.05).在不表达CEA基因的MDA-MB-231乳腺癌细胞,CEA转染诱导的CTL细胞对未分选细胞、分选的CD44+/CD24-/low亚群和非干细胞亚群的杀伤率与未转染的对照组相比,差异均无统计学意义(P＞0.05).结论:携带CEA基因rh-AAV转染DC诱导产生的抗原特异性CTL细胞可杀伤表达CEA的乳腺癌细胞,对CD44+CD24-/low乳腺癌干细胞也具有一定的杀伤活性,提示免疫治疗可能是治疗乳腺癌干细胞潜在有效的手段.     

水稻水通道蛋白OsPIP2-6的亚细胞定位研究

[目的]探讨水稻水通道蛋白OsPIP2-6的亚细胞定位.[方法]克隆了水稻中的重要通道蛋白OsPIP2-6基因,构建了瞬时表达载体,通过基因枪法在洋葱表皮中进行瞬时表达,并通过激光共聚焦显微镜进行了观察.[结果]水稻水通道蛋白OsPIP2-6主要定位在细胞质膜上,证明了OsPIP2-6是个水通道蛋白.[结论]为植物水通道蛋白的深入研究提供了理论依据.     

黏蛋白1基因磁转染树突状细胞诱导细胞毒性T细胞抗膀胱肿瘤免疫效应的研究

目的:探讨黏蛋白1(mucins 1,MUC1)基因磁转染体外人树突状细胞(dendritic cell,DC)的可行性,观察其诱导的特异性抗MUC1膀胱癌CTL的免疫效应.方法:以葡聚糖磁性纳米颗粒(DMN)作为载体,在多聚赖氨酸(PLL)的辅助下,通过静电作用结合MUC1基因的真核表达载体pEGFP-C1-MUC1,在钕-铁-硼稀土强磁块的磁场作用下转染DC,荧光显微镜下观察及流式细胞术检测转染效率,并用RT-PCR法检测转基因DC中MUC1基因的表达;再将转染MUC1基因的DC与自体T细胞共培养,并分别用乳酸脱氢酶释放法检测所致敏的细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)对MUCI特异性抗膀胱癌(膀胱肿瘤BIU87细胞系)的杀伤活性,用透射电镜观察CTL诱导靶细胞凋亡情况;ELISA法测定MUC1基因修饰后的DC刺激自体T细胞分泌IFN-γ的能力.结果:pEGFP-C1-MUC1转染效率为10%左右,荧光显微镜下可观察到明显绿色荧光蛋白的表达,RT-PCR法可检测到MUC1条带,转染MUC1基因的DC与自体T细胞混合培养后能诱导出MUC1特异性的CTL,对BIU87细胞的杀伤实验表明T-DC-MUC1的杀伤活性显著高于对照组T-DC-pEGFP-C1和T-DC诱导的CTL(P均<0.05);在透射电镜下也可观察到部分BIU87膀胱肿瘤细胞出现了细胞核核仁消失,染色质浓集于核膜周围等早期凋亡表现;基因修饰后的DC能刺激自体T细胞分泌高水平的IFN-γ,明显高于未转染的DC(P<0.05).结论:葡聚糖磁性纳米颗粒在同定磁场的作用下成功将MUC1基因转入DC,并可有效诱导出特异性抗MUC1膀胱癌的细胞毒性T细胞.     

Cre mutants with altered DNA binding properties

The recombinase Cre of bacteriophage P1 is a member of the family of site-specific recombinases and integrases that catalyze inter- and intramolecular DNA rearrangements. To understand how this protein specifically recognizes its target sequence, we constructed Cre mutants with amino acid substitutions in different positions of the presumptive DNA binding region. Here we present the results of in vitro DNA binding and in vivo recombination experiments with these Cre mutants. Most substitutions of presumptive DNA-binding amino acids in in vitro tests resulted either in the loss of target binding or in a broadening of target recognition specificity. Of the mutations resulting in a broadening of target specificity, one, N317A, results in a reduced recombination efficiency with the wild-type loxP target but recombines, in contrast to wild-type Cre, in in vivo experiments, with a symmetric variant of the wild-type target sequence. This target variant differs from wild-type loxP by the symmetric C to A replacement in position 6 of the inverted repeats. We propose a common multihelical DNA binding motif for the family of integrases and recombinases. This model implies a major structural rearrangement for the DNA binding region of lambda integrase, analogous to the structural rearrangements of the DNA binding motifs of other proteins when contacting their target DNA.     

Inducible gene targeting in mice using the Cre/lox system

We have exploited solvent perturbation to probe the coupling of Ca2+ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4 degrees C, the glycol had little effect on the myofibrillar ATPase at low [Ca2+], but at high [Ca2+] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca2+) was reduced only 3-4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca2+. Further experiments revealed that in 40% ethylene glycol, Ca2+ does initiate shortening but only with the aid of strong interactions and at temperatures above 15 degrees C. This confirms that in the organized and intact myofibril, Ca2+ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1.     

"Cre"-ating mouse mutants-a meeting review on conditional mouse genetics

BACKGROUND: The antireflux capacity of various gastric fundoplications combines the creation of a valve (flapper or nipple) with recreation of a sharp cardioesophageal angle. Experimental comparison of valve competency and appropriate valve geometry is incomplete despite wide application of these techniques. Our primary aim was to compare the competency of several antireflux valves in explanted cadaver stomachs. Our secondary aim was to understand better the geometry of the gastric fundus in empty and full stomachs. METHODS: Stomachs with 6-8 cm of distal esophagus were harvested from 18 fresh cadavers. With the stomach empty, the greater and lesser curvature length and the transverse dimensions of the anterior and posterior surface of the stomach in the fundus, body, and antrum were measured. The pylorus was tied off over a catheter; the stomachs were inflated with water; and reflux occurred. Intragastric pressure was measured during inflation with a needle inserted in the side of the stomach. A clamp was then placed on the esophagus, and the stomach was inflated to a pressure of 10 mmHg. Gastric measurements were recalculated in the distended stomach. The stomachs were deflated, the clamp removed, and a 2-cm Nissen fundoplication as well as 270 degrees and 180 degrees posterior fundoplications were performed over a 60 Fr dilator. The stomachs were reinflated while the pressure was transduced. The inflation was stopped when reflux occurred or when the fundoplication disrupted. RESULTS: The stomachs expanded symmetrically when filled with water except for the fundus in which the anterior gastric wall lengthened by more than 100% and the posterior gastric wall lengthened by about 50%. In the untreated stomachs, reflux occurred at a pressure of 3.0 +/- 1.0 mmHg. After fundoplication, reflux never occurred, but the sutures pulled out of the stomach or esophagus at 28.6 +/- 16.8 mmHg. Posterior fundoplications refluxed water in several stomachs. CONCLUSIONS: When filled, the anterior fundus expands to a greater degree than the posterior fundus, offering more tissue for creation of floppy fundoplication. The "floppy" Nissen fundoplication is completely competent, suffering a degradation before allowing reflux. The posterior partial fundoplication is unpredictable in its competency.     

Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome

The isolation and amplification of large, predetermined segments of a genome from its host have been explored. The prototype of our approach was the excisional replication of some viruses such as the lambda-lysogen. Similar machinery was used to excise and amplify large genomic segments of Escherichia coli in its host. Two loxP sequences for a site-specific recombinase Cre, together with a conditional replication origin (pi-dependent gamma-ori), were inserted into the genome by homologous recombination at predetermined sites, 50-100 kb apart. Cre and pir200 which encodes the site-specific recombinase Cre and an ori-specific replication protein pi, respectively, were also introduced into the genome. The predetermined genomic segments flanked by the loxP sequences were excised and amplified upon induction of the cre and pir200 genes which were under the control of the tet promoter. This excised and amplified DNA could be easily purified as a large plasmid. This procedure can provide an alternative to conventional cloning methods by obtaining predetermined large genomic segments directly from the original organisms. In this study, using the Cre/loxP site-specific recombination and pi/gamma-ori replication system of plasmid R6K, a procedure was devised that could isolate a large segment of the E. coli genome and demonstrated the feasibility of the procedure by excising and amplifying the 50-kb trg-narZ and 100-kb trg-hipA regions of the E. coli W3110 genome.     

Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase

The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. When genetic modifications have been generated in the germ line, however, the resulting phenotype often only reflects the first time a gene has an influence on - or is necessary for - a particular biological process. Therefore, systems allowing conditional genetic modification have been developed (for a review, see [1]); for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyse intramolecular recombination between target recognition sequences (loxP sites) in response to ligand [2] [3] [4] [5]. Here, we assessed whether a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) could be used to modify gene activity in the mouse embryo in utero. Using the enhancer of the Wnt1 gene to restrict the expression of Cre-ERTM to the embryonic neural tube, we found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene. Induction was ligand dependent, rapid and efficient. The results demonstrate that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.     

A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor

We have developed a new ligand-dependent chimeric recombinase (Cre-GRdex) by fusing the site-specific Cre recombinase to the ligand binding domain (LBD) of a mutant human glucocorticoid receptor (GRdex). The synthetic glucocorticoid receptor (GR) ligands dexamethasone, triamcinolone acetonide and RU38486efficiently induce recombinase activity in F9 murine embryonal carcinoma cells expressing constitutively Cre-GRdex. In contrast, no recombinase activity was detected in the absence of ligand or in the presence of the natural GR ligands corticosterone, cortisol or aldosterone. Moreover, physiological concentrations of these natural GR ligands do not affect Cre-GRdexrecombinase activity induced by dexamethasone. Thus, as previously shown using Cre-oestrogen receptor (ER) fusion proteins, Cre-GRdexmight be useful for achieving loxP site-directed mutagenesis in cultured cells and spatio-temporally controlled somatic cell mutagenesis in transgenic mice.     

A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs

We report the construction of two Escherichia coli strains (294-Cre and 294-FLP) which express either Cre- or FLP-recombinase. Plasmids containing authentic recognition targets for either recombinase (loxPs or FRTs) are recombined when propagated in the appropriate strain. 294-Cre and 294-FLP thus provide a simple test for the recombination competence of constructs that are designed for use in Cre- or FLP-mediated genomic manipulations.     

Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/loxP system, we developed efficient conditional transgene activation of hepatitis C virus (HCV) cDNA (nucleotides 294-3435) in transgenic mice. Efficient recombination was observed in transgenic mouse liver upon intravenous administration of adenovirus that expresses Cre DNA recombinase. After transgene activation, most hepatocytes were stained with anti-core polyclonal antibody, and 21-, 37-, and 64-kDa proteins were detected by Western blot analysis in liver lysates using anti-core, E1, and E2 monoclonal antibodies, respectively. Serum core protein was detected in transgenic mice 7 days after transgene activation with concurrent increases in serum alanine aminotransferase levels. Subsequently, an anti-core antibody response was detected 14 days after infection. Furthermore, a CD4 and CD8 positive cell depletion assay normalized both the serum alanine aminotransferase increases and pathological changes in the liver. These results suggest that HCV proteins are not directly cytopathic and that the host immune response plays a pivotal role in HCV infection. Thus, this HCV cDNA transgenic mouse provides a powerful tool with which to investigate the immune responses and pathogenesis of HCV infection.     

Non-autonomy of AGAMOUS function in flower development: use of a Cre/loxP method for mosaic analysis in Arabidopsis

Angiosperms use a multi-layered meristem (typically L1, L2 and L3) to produce primordia that then develop into plant organs. A number of experiments show that communication between the cell layers is important for normal development. We examined whether the function of the flower developmental control gene AGAMOUS involves communication across these layers. We developed a mosaic strategy using the Cre/loxP site-specific recombinase system, and identified the sector structure for mosaics that produced mutant flowers. The major conclusions were that (1) AGAMOUS must be active in the L2 for staminoid and carpelloid tissues, (2) that AGAMOUS must be active in the L2 and the L3 for floral meristem determinacy, and (3) that epidermal cell identity can be communicated by the L2 to the L1 layer.