地高辛标记Ucp2基因RNA探针的制备和应用

目的:制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法:提取小鼠胚胎脑组织总RNA,设计引物,通过RT-PCR方法获取Ucp2基因片段,将其克隆到pGEM-T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果:成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。结论:成功制备了特异高效的地高辛标记Ucp2 RNA原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。     

以反意义RNA为探针的原位杂交组织化学方法

原位杂交组织化学方法是在组织及细胞水平上研究基因表达及调控的重要方法之一。我们利用一个由体外转录产生的与小鼠阿黑促皮原(POMC)mRNA顺序互补的反意义RNA为探针,直接在大鼠垂体的组织切片上进行杂交反应。结果表明,杂交在反意义RNA探针及POMC mRNA之间进行,并形成稳定的杂交分子。放射自显影影像显示出垂体中POMC mRNA的分布及相对含量。     

烟草花叶病毒辽宁分离物Northern杂交检测体系的建立

为了建立烟草花叶病毒辽宁分离物(TMV-LN)的高特异性、高灵敏度的分子杂交检测体系,从粗提纯的TMV-LN粒子中提取RNA,设计特异性引物通过RT-PCR扩增TMV-LN的CP和3'端非翻译区域,将片段连至p UC119载体获得重组质粒p UCTMV-PP,体外转录获得地高辛(DIG)标记的TMV-LN正义链RNA杂交检测探针,同时构建DNA检测探针作为对照。采用点印迹(Dot-blot)杂交和Northern杂交对比RNA探针和DNA探针对TMV-LN的检测特异性和灵敏性。检测结果表明,RNA探针和DNA探针在点印记杂交和Northern杂交中均表现出良好的检测特异性,RNA探针在检测灵敏度方面要略好于DNA探针,且点印迹杂交体系在病毒定性方面较为快捷,Northern杂交体系在病毒基因组RNA的定量方面具有明显优势。     

地高辛标记的黄鳝F64基因cRNA探针的制备及其应用

以黄鳝F64基因序列为模板设计引物,扩增用于c RNA探针合成的模板,构建F64/p GM-T重组质粒并线性化,利用RNA聚合酶体外转录合成正、反义c RNA探针,并对其进行地高辛标记,利用原位杂交方法检测F64基因在黄鳝性腺发育过程中的表达变化情况。结果显示,正义探针未检测到阳性信号,反义探针检测到该基因在黄鳝性腺发育早期不表达,于V期性腺开始表达。研究结果表明,体外转录法可以有效合成c RNA探针,制备的c RNA探针可以准确检测F64基因的时空表达。     

RNA原位杂交技术的一些应用技巧

目的:检测基因在动物组织或细胞中的时空表达模式。方法:转录反义RNA探针;利用RNA原位杂交技术检测人和小鼠牙原基中若干基因的表达。结果与结论:通过优化条件,转录出完整的反义RNA探针,并成功地利用RNA原位杂交技术在组织中检测到基因的表达;分析了一些在RNA原位杂交的过程中可能碰到的问题及其解决方法。     

反义RNA生物工程与防治病毒性疾病的新途径

反义RNA(antisense RNA)的同义词为干扰mRNA的互补RNA(mRNA-interfering Complemeatary RNA)简称mic RNA。反义RNA与某mRNA(正义RNA)精确互补,它能在转译水平上特异性地阻断某mRNA合成蛋白质。 反义RNA首先在自然界的细菌中被发现。Mizuno等在研究大肠杆菌外膜蛋白Omp C基因时,发现该基因是双向转录的,在Omp C基因上游逆向转录174个碱基的mic F-RNA,其中有一段序列与Omp     

糖皮质激素受体mRNA的检测

用盐酸胍法和苯酚法从大鼠肝脏分离出了完整的RNA,通过oligo(dT)纤维素亲和层析从中纯化mRNA。然后用GRcDNA探针和mRNA进行Northern印迹杂交和斑点印迹杂交,放射自显影后用光密度计对糖皮质激素受体mRNA进行定量,获得了重复性较好的结果,本方法为从基因转录水平上研究GR的变化打下了必要的基础。     

cDNA微阵列分析人apoE4转基因鼠肾脏基因表达谱的变化

目的研究人apoE4转基因鼠肾脏的基因表达谱变化.方法分别提取人apoE4转基因鼠和正常C57BL/6J小鼠的肾脏总RNA,经逆转录合成cDNA探针后分别与鼠cDNA表达点阵杂交,再用ESTblot软件进行分析,并用Northern印迹证明基因表达的改变.结果人apoE4转基因鼠肾脏中有38个基因的mRNA表达升高,22个基因的mRNA表达降低.其中血浆谷胱甘肽过氧化物酶前体、视黄酸γ受体和白介素5受体等基因的表达明显增加.B-raf原癌基因、促红细胞生成素受体、整联蛋白α4的基因表达显著降低.Northern杂交证明转基因鼠肾脏的c-Jun基因表达升高.结论人apoE4转基因鼠肾脏的c-Jun、血浆谷胱甘肽过氧化物酶前体、白介素5受体等基因的表达增加;促红细胞生成素受体、整联蛋白α4等基因的表达减少.     

甲基化寡聚核苷酸作为靶基因抑制剂的研究进展

反义RNA对靶基因的抑制作用近来越来越受到人们的重视,这是因为反义RNA能在分子水平上有效地抑制有害的靶基因表达,为发展新一代治疗药物提供了可靠的理论基础。当反义RNA和靶mRNA的起始编码区(initiation coding region)互补结合或和一个靶mRNA前体的拼接区(splicing junction)互补结合形成三螺旋结构(triple stranded complexes),则这个mRNA的翻译或mRNA前体的拼接加工将被抑制,最后阻断这些靶基因正常表达。故反义RNA既可作为遗传分析的强有力的工具又可促进发展新一代基因型治疗试剂。因此,     

反义RNA及其在植物基因工程领域的应用

随着反义RNA的发现及对其研究的深入,反义RNA技术已被广泛应用于基因调控的研究中。本介绍了反义RNA的概念,并就反义RNA的作用机理和在植物基因工程领域的应用进行了综述。其作用机理包括：在原核生物中反义RNA与引物RNA前体及mRNA分子5′的不同区域进行互补,从而抑制其复制、转录和翻译;在其核生物中反义RNA影响mRNA前体拼接、转移及mRNA分子5′和3′正常修饰。在植物基因工程领域,反义RNA主要应用于抑制果实成熟、抗病、作为反向筛选标记基因、控制花色、控制淀粉合成、控制油料种子中脂肪酸的合成、控制雄性不育等方面。     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.     

A variant estrogen receptor messenger ribonucleic acid is associated with reduced levels of estrogen binding in human mammary tumors

An analysis of the human estrogen receptor (ER) mRNA was performed on 71 human breast tumors using an RNase protection assay. Complementary DNA clones to the human estrogen receptor (lambda R8 and lambda R3) were used to generate small antisense 32P-labeled RNA molecules that were hybridized to the tumor RNA. We determined the relative amounts of ER mRNA in each tumor by measuring the amount of RNases A and T1 resistant hybrids. Moreover, because RNase A has the ability to cleave single-base mismatches within RNA/RNA duplexes, we were able to use the assay to screen for possible mutations or deletions in the ER mRNA. A significant correlation was found between the ER mRNA levels and the estrogen binding concentrations determined by a dextran-coated charcoal assay (r = 0.68; P less than 0.0001; n = 58). We also identified a subpopulation of tumors in which a mismatch in the ER mRNA was detected. This message modification, in the B region of the message, significantly correlated with low levels of estrogen binding. This result suggests that the observed B variant might lead to the production of receptors with altered properties.     

IGF—ImRNA在不同年龄鲤表达的差异和LHRH—A对IGF—ImRNA表达的影响

利用RNA酶保护法对7月龄性未成熟幼鲤和2龄性成熟鲤组织胰岛素样生长因子-I（IGF-I）mRNA的表达水平进行测定,结果表明成鱼肝和肾脏组织IGF-ImRNA的丰度显著高于幼鱼,对鲤成鱼和幼鱼腹腔注射促性腺激素释放激素类似物（LHRH-A,D-Ala^6-Pro^9-NEt-LHRH)使血清生长激素（GH）水平和肝组织IGF-ImRNA水平都显著升高,而成鱼生殖腺IGF-ImRNA的丰度比对照组显著增加,研究结果提示鲤在不同发育阶段肝组织IGF-ImRNA的丰度比对照组显著增加,研究结果提示鲤在不同发育阶段肝组织IGF-ImRNA的表达存在差别,其中2龄成鱼大于7月龄幼鱼;LHRH-A可能通过刺激垂体GH的释放间接促进肝组织IGF-ImRNA的表达,亦可能通过某种未知途径刺激生殖腺IGF-ImRNA的表达。     

Majority of RNA which co-purifies with unactivated rat hepatic glucocorticoid-receptor complexes is nonspecific and not receptor-associated.

Molybdate-stabilized, unactivated rat hepatic glucocorticoid-receptor complexes were purified by a three-step procedure which includes affinity chromatography, gel filtration and anion exchange chromatography. Following elution of unactivated steroid-receptor complexes from the final DEAE-cellulose column, RNA which remained bound to the anion exchange resin was eluted with 1 M KCl. This RNA was small and heterogeneous in size. Equivalent amounts of RNA were detected after a mock purification which was devoid of receptors, suggesting that the presence of this RNA is not dependent on that of receptors. Both a [32P]DNA complementary to the RNA eluted from DEAE-cellulose and a [32P]DNA probe synthesized from total rat liver RNA gave similar results when hybridized to total rat liver RNA. These data indicated that the RNA which co-purified with unactivated receptors through the first two steps was very similar to total RNA in overall composition. Virtually identical hybridization patterns were also detected when end-labeled probes generated from the DEAE-cellulose eluted RNA or total liver RNA were hybridized to total genomic rat DNA, suggesting that the RNA eluted from the anion exchange resin is not specific or unique. Although these results do not exclude the possibility that there could be specific RNA species associated with the unactivated glucocorticoid receptor, they do indicate that the majority of the RNA eluted from DEAE-cellulose following elution of receptor complexes appears indistinguishable from total rat liver RNA and can be detected in parallel mock purifications.     

Regulation of rat growth hormone receptor gene expression

A cDNA encoding the growth hormone (GH) receptor was cloned from rat liver. Both the nucleotide and translated amino acid sequence share greater than 70% similarity with the GH receptors from rabbit and human. An RNA probe was generated from this sequence for use in a solution hybridization assay to quantitate GH receptor mRNA expression in rat tissues. Expression was detected in 9/12 tissues examined, with the highest levels observed in the liver. Expression in liver, kidney, heart and muscle was developmentally regulated, being low at birth and rising to adult levels in 5 weeks. No difference was observed between hepatic expression in males and females, although livers from pregnant rats had elevated levels. Hypophysectomy and GH treatment did not affect hepatic GH receptor mRNA levels.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.