Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Knocking-Down Cyclin A(2) by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo. (C) 2010 Elsevier Inc. All rights reserved.

RNA干扰在抗病毒研究中的应用

国家自然科学基金海外杰出青年基金(30428024)资助

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

APOBEC3G 多重转录本的发现和部分表达特征的研究

APOBEC3G（apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G，载脂蛋白B mRNA编辑酶催化多肽样蛋白3G，简称为A3G）是2002年Sheehy 发现的天然抗病毒限制因子，代表近年来病毒研究领域的一项重大进展,不仅拓 展了对病毒宿主相互作用的认识,也为研发抗病毒药提供了新的思路和方向。不 过，A3G的结构、功能和抗病毒机制仍有许多未解的重大问题，进一步深入和系 统的研究，可能为预防和治疗HIV/AIDS及多种病毒性疾病，提供新的线索。 本研究的最初线索，来源于前期工作中的偶然发现：我们试图在一个健康个 体PBMC 中克隆A3G 编码序列，经双向测序后发现，有5 个位点属非同义突变， 造成氨基酸序列的改变。为排除个体差异并出于研究的便利，我们采用单核样细 胞系THP-1，进行多条序列的克隆和分析。在研究中，我们使用错误率仅为 1.6x10-6 的高保真酶Pfu，共克隆到23 条A3G 基因编码序列。经双向测序和对比 分析，较为意外地发现，其中20 条序列具有2 个以上的非同义突变或截断错位， 突变率高达87%，显示A3G 基因在THP-1 细胞中存在多重正常或变异的转录本。 这一现象，是否代表天然突变的普遍性以及其频度和意义，有待于进一步在多种 细胞和人群多个个体中验证和大规模的系统研究。 为建立基本的实验体系和研究手段，我们通过RT-PCR 与Western blotting， 检测了A3G 基因在9 个细胞系内的本底表达水平，并在此筛选结果的基础上， 在低本底的细胞系中，以慢病毒系统表达A3G 基因，而在高本底细胞系中，通 过siRNA 沉默A3G 基因，建立了A3G 高低表达体系，为后续研究A3G 功能提 供了方便可行的实验平台。 我们选取野生型A3G、带有3 个位点（H186R、I309T、F310S）连锁的突 变体和5 个位点（W34R、Y222H、V224G、A246V、F289L）非连锁的突变体， 构建真核表达载体，转染HEK293T 细胞，对比观察不同转录本在细胞中的表达 动态和水平。结果发现，3 个位点的突变对A3G 蛋白细胞内定位无明显影响，5 个位点的突变明显增加了单个细胞中A3G 蛋白点灶状团块的比例。并且突变位 点明显减缓了A3G 的表达速率，降低了表达水平。而连锁、非连锁以及截断错 位的突变体在抗病毒功能和结构生物学上的意义，有待于进一步的深入分析。 综上，我们发现A3G 在THP-1 细胞中存在多重转录本，并在确定细胞系A3G 本底表达的基础上，建立了A3G 体外高低表达体系，为后续我们进一步研究 A3G 的功能奠定了基础。而部分突变体在细胞内的表达趋势和细胞内定位的观 察和分析，为进一步的深入研究提供了新的线索。

贾第虫microRNA 及其靶基因的预测与验证

MicroRNAs (miRNAs)是一类长约21-25nt 的非编码小分子RNAs，通过与靶基因的互补结合在转录水平及转录后水平来负调控基因表达。人们已在众多高等多细胞生物中如人、果蝇、线虫、拟南芥等鉴定出众多microRNAs 分子。近来报道单细胞原生生物衣藻中也存在大量microRNAs。然而到目前为止，在被很多证据证实是最原始的单细胞真核生物贾第虫中却仍未有microRNAs 的报道。那么到底贾第虫这种具有特殊进化地位的单细胞原生动物是否存在有microRNAs 呢？如果存在的话，其microRNAs 的特点是什么？与高等多细胞生物及单细胞衣藻的 microRNA 相比又有何异同点呢？贾第虫的microRNAs 是否与其致病性相关呢？已有研究表明，贾第虫基因组中存在与RNAi 相关的Argonaute（AGO）家族蛋白和Dicer 酶。有意思的是，这些与siRNA 引起RNAi 作用关键的蛋白AGO 和Dicer 同样也是miRNA 系统的关键成份，这就提示我们在贾第虫中很有可能也存在有miRNA 并发挥功能。有研究发现在贾第虫基因组中存在大量的非编码转录物，这些大量的非编码转录物中，是否都是后来所认为的为双向启动子转录有用基因时的副产物，还是也存在一些起调控作用的RNA 分子（如miRNAs 等），需要进一步的研究。本文利用生物信息学的手段，依据miRNAs 的生物学特征，结合多种计算机预测的方法，在贾第虫基因组中筛选可能的microRNAs 分子，结果共鉴定出50 个miRNAs 候选分子，这50 个可能的贾第虫miRNAs 不具有保守性，在已知的其他物种的miRNAs 中找不到同源物。用这50 个microRNAs BLASTN 贾第虫的蛋白质编码序列及其相邻5’端和3’端各200bp 的序列，来寻找这些microRNAs 所调控的靶基因。结果表明，寻找到的贾第虫miRNA 的靶基因除很大一部分未知功能的蛋白外，还包括了很多涉及不同功能的蛋白，如VSP 蛋白（various surface proteins）这样一类表面抗原蛋白，提示我们贾第虫miRNA 可能与其致病性相关。接下来我们对其中14 个预测的贾第虫microRNAs 进行了RT-PCR 检测并克隆测序，结果表明gla-mir-6, gla-mir-35 在贾第虫滋养体细胞中稳定表达。我们的研究第一次用生物信息学结合实验的方法在贾第虫寻找到了microRNAs，为下一步深入研究这些microRNAs 在贾第虫中的功能提供了可能。

RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

Survivin表达对HepG2细胞高LET射线辐射敏感性的影响

<正>Survivin是1997年发现的凋亡抑制蛋白家族(IAPs)的成员,它特异性的表达于大多数的恶性肿瘤细胞中,而在正常组织中检测不到,具有组织特异性。以前的研究表

抑制survivin表达增强人肝癌HepG2细胞对高线性能量转移射线的辐射敏感性；Inhibiting Survivin Expression Increases The Radiosensitivity of Human Hepatoma HepG2 Cells to High-LET Radiation

探讨了肿瘤细胞中survivin的表达对高线性能量转移(LET)射线辐射敏感性的影响.根据Gen Bank提供的survivin序列,合成特异性survivin-siRNA寡核苷酸,转染人肝癌HepG2细胞,抑制survivin的表达.发现siRNA转染后诱导了HepG2细胞G2/M期阻滞,增加了自发性和辐射诱导的细胞凋亡.在高线性能量转移(LET)碳离子辐照后,siRNA转染细胞的克隆存活率明显下降.这些结果表明survivin表达是HepG2细胞产生对高LET射线辐射抗性的关键因素.