U6和H1双启动子载体用于RNAi的实验研究

为建立一条简便、有效的构建哺乳动物细胞随机RNAi文库技术路线 ,首先通过PCR将 1 9～ 2 1ntsiRNA编码序列及终止信号等元件引入U6启动子下游 ,再将该扩增产物定向克隆至H1启动子的下游。构建的双启动子载体中 ,U6和H1相对排列 ,均以其间插入的靶序列为模板 ,分别转录出其正义和反义链 ,形成功能性siRNA。以EGFP和bcl-2为靶基因的实验表明 ,该方法构建的载体可有效地干扰相应基因的表达。     

小鼠RNA干扰基因RelA慢病毒载体的构建与鉴定

目的：构建小鼠RelA 基因的RNA 干扰慢病毒载体,转染小鼠成骨样细胞并鉴定。方法：针对小鼠RelA 基因序列,设计特异 性的shRNA 序列,应用基因重组技术插入慢病毒载体GV-248。得到的重组质粒转化感受态大肠杆菌DH5-alpha,筛选得到阳性克隆 并扩大培养。所得质粒进行测序分析确定载体构建成功。重组质粒载体及包装辅助质粒转染293T 细胞,得到目的病毒并测定相 应病毒滴度。慢病毒转染MC3T3-E1 细胞后,Real-time PCR 及Western blot 检测MC3T3-E1 细胞RelA 基因及成骨相关基因 ALP、OCN、RANKL的表达。结果：成功构建小鼠RelA 基因的RNA干扰慢病毒载体,感染MC3T3-E1 细胞后,RelA 基因的表达 明显受到抑制,同时RANKL基因表达水平明显下降,ALP、OCN基因表达水平明显上升。结论：成功构建了小鼠RelA 基因的 RNA 干扰慢病毒载体。当小鼠成骨细胞RelA基因表达被干扰,NF-资B 通路被抑制后,小鼠成骨细胞成骨相关基因ALP、OCN的 表达明显上升,成骨功能增强;同时RANKL 的表达明显下降,其介导的破骨细胞骨吸收功能减弱。     

乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒的构建及鉴定

目的：构建乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。方法：乙酰肝素酶cDNA全长扩增和最佳siRNA干扰片段筛选分别采用PCR和Real-time PCR方法,慢病毒系统载体分别使用pWPI和siRNA pSico系统,采用限制性内切酶快速连接方法联接目的基因和最佳最佳siRNA干扰片段,表达载体鉴定均采用核苷酸序列测定,HPSE重组慢病毒表达质粒和siRNA片段细胞转染采用脂质体转染法。结果：成功扩增乙酰肝素酶全长并连接入pWPI载体构建成重组表达载体HPSE-pWPI,重组质粒测序结果显与HPSE基因的同源性达99%。转染293T后有HPSE基因的表达。筛选出最佳siRNA干扰片段为HPSE-1222并成功插入pSico载体,构建成重组表达载体HPSE-siRNA pSico,重组载体测序显示与构建的shRNA结构序列完全一致。结论：成功采用慢病毒载体系统构建了乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。     

慢病毒载体介导的层黏连蛋白受体稳定抑制细胞株的建立

目的:构建靶向层黏连蛋白受体(LR)基因的小发卡RNA(sh RNA)慢病毒表达载体,鉴定其对LR的抑制效果,并筛选LR稳定抑制的He La细胞株。方法:设计针对LR的sh RNA序列,将此序列和H1启动子克隆入含有EGFP报告基因的p Lenti6/v5慢病毒表达载体,通过病毒包装、细胞感染、抗生素筛选获得稳定细胞株,用real-time PCR和Western印迹检测筛选得到的稳定细胞株中LR的表达水平。结果和结论:构建了含有LR靶向sh RNA的慢病毒表达载体,包装成病毒后感染He La细胞,经抗生素筛选后获得了稳定抑制LR的细胞株;筛选后的细胞均可观察到报告基因EGFP的表达;经m RNA和蛋白水平检测,LR-sh6和LR-sh7均可显著抑制He La细胞株中LR的表达。     

玉米淀粉分支酶基因的克隆与RNAi表达载体的构建

利用网络数据库http://jura.wi.mit.edu/bioc/siRNAext/,确定玉米淀粉分支酶SBEⅡb的部分基因序列作为RNAi的目标序列,通过RT-PCR方法从玉米自交系‘综31’中分别成功克隆出了玉米淀粉分支酶sbeⅡb基因的部分序列及其启动子序列,从大肠杆菌中成功克隆了木糖异构酶基因xylA作为筛选标记,构建了含有35S驱动的木糖异构酶基因和sbeⅡb启动子驱动的含有sbeⅡb目标序列的反向重复序列的表达载体pBAC413,并对玉米自交系实施转化。经PCR检测转基因玉米再生植株,初步判定目的序列已经成功整合到玉米再生植株的基因组中。     

腺病毒载体介导的GFP基因在鸭胚成纤维细胞中的表达及RNA干扰

目的：建立在鸭胚成纤维细胞（DEF）中进行RNA干扰（RNAi）的技术平台,为鸭基因组功能的研究提供新的技术手段。方法：以绿色荧光蛋白（GFP）基因为报告基因,脂质体转染化学合成的GFP特异小干扰RNA（GFP-siRNA）,用流式细胞仪测定GFP-siRNA对重组腺病毒（Adv-GFP）介导的GFP基因在DEF中表达的干扰效果。结果：200MOI（感染复数）Adv-GFP介导的GFP基因在DEF中表达效率最高,为31．20％±3．1l％,对DEF的活力无明显影响;GFP-siRNA能有效干扰GFP基因在DEF中的表达,相对抑制率为98．56％。结论：在DEF中进行RNAi是可行的,Adv-GFP是介导外源基因在DEF中表达较为理想的载体;首次建立了在DEF中进行RNAi的技术平台,为鸭基因组的功能等研究提供了新的技术手段。     

噬菌体phi29 pRNA载体在RNA干扰中的初步应用研究

RNA干扰是在细胞胞质中双链RNA(dsR-NA)介导的序列特异性mRNA的降解[1]。这个过程是由21~25个被称为小干扰RNA(si RNA)形成的dsRNA完成[2]。目前,这一技术已经广泛应用于研究基因的功能,病毒感染治疗等方面。但是,si RNA在体内容易降解,干扰作用持续的时间不长。新的研究表明枯     

RNA干扰机制及其应用研究进展

2006年诺贝尔生理学或医学奖,授予给了美国科学家Andrew Z.Fire和Craig C.Mello以表彰他们发现了RNA 干扰(RNAinterference RNAi)现象,使人们在基因治疗传染性、恶性肿瘤等危重疾病领域取得了突破性的发展.本文主要综述了RNA干扰系统的组成、分子机制、作用特点及其在探索基因功能、传染性、恶性肿瘤的基因治疗和药物研发等四个方面的应用.     

可诱导慢病毒载体的优化策略及应用

以1型人免疫缺陷病毒(HIV-1)为基础构建的慢病毒载体具有可感染非分裂细胞、免疫反应小、携带的基因片段容量大和可整合进宿主基因组而长期表达等优点,因而成为最理想的基因转移载体之一。可诱导慢病毒载体介导的可诱导基因表达系统能够有效控制目的基因表达,扩大了慢病毒载体的临床应用潜能,成为很有前景的基因治疗载体。主要介绍带有四环素和其他几种诱导系统的可调控性慢病毒载体及其改进,以及可诱导慢病毒载体在RNA干扰中的应用。     

农杆菌介导淀粉分支酶基因RNAi片段转化玉米的研究

以玉米自交系“178”和“R18红”的胚性愈伤组织为材料,通过愈伤组织对潮霉素的敏感性实验,确定了潮霉素15 mg/L~25 mg/L为愈伤组织适宜的选择压。利用农杆菌(Agrobacterium tum efaciens)介导将淀粉分支酶基因RNA干涉表达载体转入玉米自交系中,并对农杆菌转化系统的条件进行研究。结果表明:在感染液和共培培养基中分别都加入100μm ol/L乙酰丁香酮和50 mg/L抗坏血酸,农杆菌LBA4404的菌液OD600为0.6、侵染时间20 m in为农杆菌转化的最适条件。对转化的愈伤组织分化诱导出苗后进行PCR检测,证明外源目的基因已整合到玉米基因组中,转化率最高达到2.4%。     

可诱导表达Mesp1的慢病毒载体构建及P19细胞稳定表达系的建立

Mesp1属于b HLH转录因子家族,对早期心脏中胚层的形成十分重要。为研究Mesp1在心脏发育过程中的功能,本文构建了可诱导表达Mesp1的慢病毒载体,转染进P19细胞,经嘌呤霉素筛选并扩大培养后,成功建立起经强力霉素诱导后可稳定过表达Mesp1基因的细胞系P19-Mesp1,为研究Mesp1心脏发育相关功能提供有用的细胞研究模型。     

In vivo longitudinal imaging of RNA interference‐induced endocrine therapy resistance in breast cancer

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor‐positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence‐labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)‐regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox‐induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor‐bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen‐treated control group. Longitudinal imaging was performed by a home‐built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper‐measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e‐7) correlated with the tumor size progression for the mice with shRNA‐induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.      

Reversible Suppression of Cyclooxygenase 2 (COX-2) Expression In Vivo by Inducible RNA Interference

Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3′untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.     

p300基因小干扰RNA慢病毒载体的构建及其对乳腺癌ZR75-1细胞的影响

目的:构建p300基因的小干扰RNA(siRNA)慢病毒表达载体,检测其对p300蛋白表达的干扰,以及对乳腺癌细胞ZR75-1生长的影响。方法:利用RNA干扰(RNAi)技术设计并合成针对p300基因的siRNA,并将其克隆到siRNA表达载体pSIH-H1上,经酶切和测序验证,用293T人胚肾细胞包装p300 siRNA慢病毒,感染乳腺癌细胞ZR75-1,建立敲低p300表达的稳定细胞株,通过实时定量PCR和Western印迹检验RNAi的干扰效果,利用细胞生长实验检测p300 siRNA对ZR75-1细胞生长的影响。结果:酶切和测序证明构建了p300 siRNA真核表达载体;实时定量PCR和Western印迹证明构建的siRNA能有效抑制p300基因的表达,并建立了敲低p300表达的稳定细胞株;细胞生长实验证实p300 siRNA可有效促进ZR75-1细胞的生长。结论:构建了p300基因的siRNA真核表达载体,转染细胞后能有效抑制p300基因的表达,且能促进ZR75-1细胞的生长,表明构建的p300 siRNA具有功能。     

携带三种报告基因慢病毒载体的构建及其实验研究

慢病毒是逆转录病毒科的一个属.它具有可以感染分裂期及非分裂期细胞、容纳外源性目的基因片段大、目的基因表达时间较长、免疫反应小等诸多优点,因此成为运载目的基因的理想载体而得到广泛的应用.构建了带有3种报告基因即红色荧光蛋白(mCherry)、荧光素酶基因(luciferase)及绿色荧光蛋白(eGFP)的慢病毒载体,其中...     

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers. We show that shRNAs, which target the vector genomic RNA, strongly reduced lentiviral vector titers but inhibition of the RNAi pathway via saturation could rescue vector production. The presence of miRNAs in the vector RNA genome (sense orientation) results in a minor titer reduction due to Drosha processing. A major cause for titer reduction of miRNA vectors is due to incompatibility of the cytomegalovirus promoter with the lentiviral vector system. Replacement of this promoter with an inducible promoter resulted in an almost complete restoration of the vector titer. We also showed that antisense poly(A) signal sequences can have a dramatic effect on the vector titer. These results show that not all sequences are compatible with the lentiviral vector system and that care should be taken in the design of lentiviral vectors encoding RNAi inducers.     

结缔组织生长因子基因RNA干扰复制缺陷型腺病毒载体的构建及鉴定

目的：构建能介导结缔组织生长因子（CTGF）基因RNA干扰的复制缺陷型腺病毒表达载体。方法：以大鼠CTGF基因为靶序列,设计并合成含编码短发夹RNA序列的寡核苷酸,构建腺病毒穿梭质粒P-shuttle-CTGF,酶切及测序分析正确后,与腺病毒骨架质粒pAdEasy-1共转染AD-293细胞,进行病毒包装,得到腺病毒载体Ad．H1-CTGF,用该载体感染HSC-T6细胞,观察其对CTGF基因表达抑制的效果。结果：构建的腺病毒穿梭质粒p-shuttle-CTGF经酶切、测序分析证实正确;包装的病毒载体滴度为4×1010PFU／mL,感染HSC-T6细胞后,Western印迹证实CTGF表达显著减少。结论：构建的腺病毒载体Ad．H1-CTGF可有效抑制HSC-T6中CTGF的表达,为抗纤维化研究提供了有力的工具。     

纳米载体介导的植物遗传转化研究现状和前景

高效遗传转化技术是植物重要性状功能基因鉴定的前提和转基因育种的基础.随着纳米生物技术的发展,以纳米载体介导的植物转基因技术已显示出巨大的应用潜力.综述了国内外应用于植物纳米载体的类型、与外源基因的结合方式以及传输细胞的原理,重点阐述了影响纳米基因载体性能与转化效率的重要因素,以及纳米载体介导外源基因转化植物细胞的方法,...