哺乳动物转基因载体——piggyBac转座子

piggyBac转座子具有识别位点特异性5'-TTAA-3',剪切和插入都不留下印迹,且高效转座。基于piggyBac转座子的特性,借助载体系统,已应用于哺乳动物转基因的研究。此外,piggyBac转座子还应用于基因诱导突变和基因治疗等领域。RNAi技术作为基因功能研究的一个工具,在应用于克服畜牧业中家畜的生产繁殖障碍等方面已取得一定研究成果。本文对piggyBac转座子的结构和特性、转座机制及影响因素和在动物中应用等方面进行总结,为RNAi技术借助piggyBac转座子用于哺乳动物转基因提供理论依据。     

piggyBac转座子应用研究进展

piggyBac转座子是来源于鳞翅目昆虫粉纹夜蛾(Trichoplusia ni)的DNA型转座子。目前piggyBac转座子已成为在昆虫中应用最广泛的转座子之一。近年来国内外研究者更将其应用领域拓宽到了鱼类,寄生虫和哺乳动物等转基因研究中。随着对piggyBac转座子功能和分布的深入认识,piggyBac转座子将更多应用于基因功能研究,基因治疗,转座子介导的细胞系基因诱变及基因工程蛋白表达等基础研究和应用研究中。     

昆虫转座子及在家蚕中的应用

概述了近年来广泛应用于昆虫的若干转座子及其基本构造和应用;着重讨论了piggyBac转座子的构造和转座机制及所作成的转基因昆虫种类及标记基因:同时列举了在家蚕转基因中的实例.     

piggyBac转座子在畜牧兽医科学中应用研究进展

piggyBac（PB）转座子已被证明是一种高效的非病毒基因工程操作工具,现广泛用于基因操作和转基因动物研究中。借助PB转座子已获得转基因小鼠、鸡、猪、山羊等动物。文中重点就PB转座子在畜牧兽医科学研究中的应用进行综述。     

禽类转座子的研究进展

转座子作为一段可移动的DNA序列,对于生物体的基因组进化和物种分化发挥着重要作用。近年来,转座子在生物突变体制备、转基因研究和基因治疗等领域展现出了非常大的潜能。然而目前关于动物转座子的研究主要集中在哺乳动物,禽类转座子的相关研究较少。禽类基因组内的转座子元件具有含量少、多样性低等特点,同时禽类转座子在基因组中的分布也与哺乳动物有很大差异。文章综述了各类转座元件的结构特征和挖掘方法以及禽类基因组中转座子的种类和特点,并对禽类基因组内转座元件的研究进展和存在的问题进行阐述,旨在提高禽类转座子研究的重视程度,为禽类转座子的后续研究提供理论参考。     

mariner 转座子及其在昆虫种系转化中的应用

转座子是不需要同源重组便能在基因组内和基因组间移动的遗传学顺式元件.mariner是最简单的真核转座子,在昆虫中广泛分布.mariner转座子的转座作用只与转座酶有关,是一种很有发展前景的转基因载体,可以实现异源昆虫间的基因转移,产生稳定遗传的转基因品系.     

地熊蜂基因组中具有潜在活性的转座子鉴定

转座因子(TEs)是真核生物基因组的主要序列成分,对真核生物基因组的结构、功能及进化具有重大影响。近年来,虽然有关昆虫转座子的研究在不断增加,并且科学家成功地应用转座子进行了功能基因的挖掘,但相关研究主要集中于双翅目的果蝇中。本研究利用生物信息学方法,基于de novo预测和结构预测两种策略,对地熊蜂参考基因组中的转座子进行了详细鉴定、分类和注释,并鉴定出潜在活跃的转座子。结果显示:虽然转座子序列仅占地熊蜂基因组全部序列的3.74%,但其超家族种类繁多,鉴定出的33282个转座子分属于22个超家族。本研究鉴定出2种具有潜在活性的MITE转座子,它们很可能正在地熊蜂基因组中发生转座。本研究为利用活跃的转座子创制地熊蜂的突变体库进而挖掘熊蜂的功能基因奠定了重要基础。     

慢病毒载体及慢病毒介导的RNA干扰

慢病毒载体是高效的基因转导工具,能将外源基因序列稳定导入分裂期细胞和非分裂期细胞.将慢病毒载体与RNA干扰结合能在哺乳动物的各类细胞中特异性抑制同源基因的表达,它将是基因功能研究和基因治疗的有力手段.应用慢病毒载体进行转基因动物的制备,转基因的效率将得到显著提高.慢病毒介导的RNA干扰具有高效、稳定、特异性强的特点,它能在哺乳动物的各类细胞、多种疾病的离体细胞中实现稳定的RNA干扰.该技术被广泛用于基因功能的研究,在疾病的基因治疗上也具有良好的前景.     

慢病毒载体及慢病毒介导的RNA干扰

慢病毒载体是高效的基因转导工具,能将外源基因序列稳定导入分裂期细胞和非分裂期细胞.将慢病毒载体与RNA干扰结合能在哺乳动物的各类细胞中特异性抑制同源基因的表达,它将是基因功能研究和基因治疗的有力手段.应用慢病毒栽体进行转基因动物的制备,转基因的效率将得到显著提高.慢病毒介导的RNA干扰具有高效、稳定、特异性强的特点,它能在哺乳动物的各类细胞、多种疾病的离体细胞中实现稳定的RNA干扰.该技术被广泛用于基因功能的研究,在疾病的基因治疗上也具有良好的前景.     

动物基因组中反转录转座子的研究进展

反转录转座子 (retrotransposon)是一类通过RNA实现转座的遗传因子 ,以多拷贝形式广泛存在于真核生物基因组中。由其产生的转座会导致基因组序列的删除、扩增、移位、断裂、重组等现象 ,从某种意义上说反转录转座子是生物遗传多样性产生的一个重要因素     

转基因表达的调控方法

转基因动物技术作为一种研究基因功能的技术体系 ,在生命科学研究领域有着广泛的应用前景。有效地使精确的遗传基因修饰在动物体内得到表达并实现世代间的传递是转基因技术的关键。近年来由于转座子、逆转录病毒的运用以及采用加入或去除某些基因的体细胞的克隆技术的发展 ,不同物种转基因的培育方法日趋简便。 Cre-L ox P系统越来越多地用于从基因组中去除特定的序列或靶向整合外源DNA。四环素等系统已被证实可获得确切的转基因表达。具有反式显性阴性效应的与 DNA形成三链螺旋的 RNA、反义 RNA(包括 :含 RNA干预和核酶的双链 RNA)以及蛋白质的表达均被证实可特异性地抑制宿主或病毒基因的表达。文章综述了转基因的概念、表达及调控转基因表达的常用方法     

表达靶向禽流感病毒多靶点miRNA重组慢病毒的制备及病毒感染效率检测

慢病毒介导的RNAi具有转移基因效率高,作用持久稳定等特点,成为基因治疗和基因功能研究的重要工具。本试验中将在细胞水平验证可以抑制禽流感病毒(AIV)PA、NP和PB2基因表达的miRNA克隆到pcDNATM6.2-GW/EmGFP-miR载体,构建多靶点miRNA表达载体(pcDNA6.2/PA+NP+PB2);鉴定正确后通过BP/LR重组反应将GFP和靶向AIV串联miRNA转座到慢病毒表达载体pLenti6/DEST,命名为pLenti6/PA+NP+PB2;鉴定正确后与辅助包装质粒共转染293FT,72h收集细胞上清进行病毒浓缩;采用梯度稀释法和real-timePCR法测定病毒滴度;通过感染MDCK细胞、CEF细胞及猪胎儿成纤维细胞,评价重组慢病毒的感染效率。结果,酶切和测序表明pcDNA6.2/PA+NP+PB2和pLenti6/PA+NP+PB2构建成功;浓缩后梯度稀释法检测病毒滴度为4×107TU/mL,real-timePCR法检测病毒滴度为1×108TU/mL;病毒感染MDCK细胞和猪胎儿成纤维细胞的感染效率显著高于CEF细胞的感染效率。结果表明,我们成功制备了表达靶向AIV多靶点miRNA重组慢病毒,并发现以VSVG替代了env囊膜后慢病毒对CEF细胞敏感性较低,为进一步研究AIV的防控和慢病毒介导的抗AIV转基因动物模型奠定了基础。     

宿主蛋白ANP32A对流感病毒功能影响的研究进展

流感病毒是一类危害人和动物健康的RNA病毒,其在宿主细胞内的有效复制离不开宿主蛋白酸性核磷蛋白32家族成员A （ANP32A）和病毒RNA聚合酶的协助和支持。病毒RNA聚合酶由3种蛋白PB1、PB2和PA组成,且ANP32A与病毒RNA聚合酶的最强相互作用需要这3种蛋白的共同参与。ANP32A是酸性富含亮氨酸的核磷蛋白32（ANP32）家族成员,其被确认为支持细胞核中病毒RNA聚合酶活性的关键宿主因子,对流感病毒的复制具有重要的作用。ANP32A的物种特异性差异决定了病毒RNA聚合酶的宿主范围：独特的33个氨基酸序列存在于禽类ANP32A （avANP32A）,而在哺乳动物ANP32A中缺乏此氨基酸序列。avANP32A中特有的33个氨基酸序列能增强ANP32A的功能,从而增加禽源特征流感病毒聚合酶活性。禽流感病毒（Avian influenza virus,AIV）不能有效利用较短的ANP32A （即缺乏独特33个氨基酸序列的ANP32A）,因而哺乳动物ANP32A无法支持禽源特征聚合酶活性,然而在人ANP32A （huANP32A）中插入这33个氨基酸能促进其对AIV聚合酶的支持作用。此外,流感病毒的适应性突变也能增强AIV在哺乳动物中的传播力和致病性。AIV适应哺乳动物时往往会发生E627K突变,以增强其在哺乳动物中的复制能力。作者主要介绍了宿主蛋白ANP32A对流感病毒复制、转录的影响和流感病毒发生适应性突变的作用机制,简要论述了ANP32A与聚合酶的相互作用对流感病毒跨物种感染的分子机制。     

Mechanical probes can predict tenderness of cooked beef longissimus using uncooked measurements

Two experiments were conducted to determine the effectiveness of using mechanical probes and objective color measurement on beef LM to predict cooked tenderness. In Exp. 1, sharp needle (SN), sharp blade (SB), blunt needle (BN), blunt blade (BB), and plumb bob (PB) probes were used to measure uncooked LM (n = 29) at 2 d postmortem in both a perpendicular and parallel orientation to the long axis of the strip loin. Additionally, instrumental color measurements were measured on uncooked muscle at 2 d postmortem. Steaks for trained sensory panel (TSP) and Warner-Bratzler shear force (WBSF) measurements were aged 14 d postmortem before cooking. Probe measurements taken perpendicular to the long axis of the LM were not correlated (P = 0.22 to 0.82) to TSP tenderness. Probe measurements (BB, BN, SN, SB, and PB) taken parallel to the long axis were correlated to TSP tenderness (r = -0.57, -0.40, -0.77, -0.52, and -0.53, respectively). A regression equation using the SN probe to predict TSP tenderness had a R2 value of 0.74. The SB probe combined with L* accounted for 45% of the variation in TSP tenderness, whereas the PB probe combined with L* accounted for 56% of the variation in TSP tenderness. A second experiment (n = 24) was conducted using the SN, SB, and PB probes on uncooked sections at 2 d and on cooked steaks at 14 d postmortem. Probe measurements on cooked steaks were not correlated to TSP tenderness. New regression equations were calculated using the probe measurements on uncooked steaks from both experiments. Prediction equations formulated with L* values and either SN, SB, or PB probes accounted for 49, 50, and 47% of the variability in TSP tenderness scores, respectively. An equation using WBSF of cooked steaks to predict TSP tenderness had an R2 of 0.58. Of the steaks predicted to be tender (predicted tenderness > 5.0) by the equations using the SN, SB, and PB probes on uncooked steaks and WBSF on cooked steaks, 85, 88, 80, and 84%, respectively, were actually tender (TSP tenderness > 5.0). Mechanical probe measurements of uncooked steaks at 2 d postmortem can potentially classify strip loins into groups based on tenderness, as well as WBSF measurements, which are more costly and time consuming.     

锌指核酸酶技术在动物转基因中的研究进展

Zinc-finger nucleases (ZFNs) are engineered site-specific DNA cleavage enzymes that may be designed to recognize long target sites and thus cut DNA with high specificity. ZFNs mediate permanent and targeted genetic alteration via induction of a double-strand break at a specific genomic site. Compared to conventional homology-based gene targeting, ZFNs can increase the targeting rate by up to 100000-fold; gene disruption via mutagenic DNA repair is similarly efficient. The utility of ZFNs has been shown in many organisms, including insects, amphibians, plants, nematodes, and several mammals, including humans. This broad range of tractable species renders ZFNs a useful tool for improving the understanding of complex physiological systems, to produce transgenic animals, cell lines, and plants, and to treat human disease. This review summarized recent advances in the understanding of role and function of ZFNs and described the utility of ZFNs for mammalian transgenesis.     

Effect of polymyxin B and environmental conditions on isolation of Brucella species and the vaccine strain RB51

Brucella are resistant to polymyxin B (PB), but their relative susceptibility to PB and its derivative, colistin (COL) has not been rigorously or systematically studied. Comparative susceptibility of Brucella reference strains, vaccine strain RB51, and Brucella isolates from marine mammals to these two cationic peptides were determined by Etest. Vast differences among Brucella species were found in susceptibility to both PB and COL. Brucella demonstrated similar pattern of relative susceptibility to PB as that of COL, but they were less susceptible to COL. Both B. melitensis and B. suis were the least susceptible to polymyxins and rough strains were more susceptible to both PB and COL than the smooth except for the BvrR mutant. Strains were generally less susceptible to PB when cultured in CO(2) rather than ambient air; some became more susceptible in acidified medium. Results show that environment cultural conditions must be considered when selecting for CO(2)-independent strains of Brucella especially the vaccine strain RB51 on selective media containing PB. Our observations extend basic knowledge of the differential resistance of Brucella to polymyxins.     

RNA干涉与基因沉默的研究进展

RNAi是指外源dsRNA引发身体内的基因的同源序列降解,从而表现出基因转录后的沉默后现象,到目前为止在真菌、拟南芥、线虫、锥虫、水螅、涡虫、果蝇、斑马鱼、小鼠等真核生物中都发现存在这一基因沉默机制。研究表明,RNA干涉与植物中的共抑制、真菌中的基因压制熏很可能具有共同的基本分子机制。它可以用于功能基因组学研究,也可用于克服转基因生物的基因沉默现象,使外源基因在遗传改良生物中能更好地表达,还用于基因治疗,抑制有害基因的表达等。     

转人乳铁蛋白基因奶牛多重PCR检测方法的研究

本研究旨在建立转基因奶牛多重PCR快速检测方法,为转基因动物及产品出入境检测技术平台的建立提供技术支持,并为转基因动物及产品检测技术标准的制定提供参考。根据牛物种特异性基因(mtDNA)设计奶牛内源基因引物,根据外源基因人乳铁蛋白基因(human lactoferrin,hLF)和新霉素磷酸转移酶基因(NPT Ⅱ)设计特异性引物,优化反应条件,建立转基因奶牛多重PCR检测方法。本方法敏感、快速、特异,一个反应可以检测多个基因片段,可有效用于转基因奶牛外源基因的检测。     

The role of p38 mitogen-activated kinase (MAPK) in the mechanism regulating cyclooxygenase gene expression in equine leukocytes

The goal of this study was to define the role for p38 mitogen-activated kinase (MAPK) in the signaling mechanism regulating pro-inflammatory cyclooxygenase (COX) gene expression in lipopolysaccharide (LPS)-activated equine leukocytes for the purposes of identifying novel targets for anti-inflammatory therapy in endotoxemic horses. The p38 MAPK has been shown to positively regulate inflammatory gene expression in human leukocytes and can be activated by a variety of stimuli including LPS, TNF-alpha, and IL-1. Activation-associated phosphorylated p38 MAPK has been implicated in the up-regulation of several inflammatory genes, including COX-2 which ultimately results in the production of prostanoids that are responsible for the pathophysiology associated with endotoxemia. Our hypothesis is that activation of p38 MAPK is essential for LPS-induced COX-2 expression in equine peripheral blood leukocytes. We tested our hypothesis by investigating the effects of the specific p38 MAPK inhibitors SB203580 and SB202190 on LPS-induced COX-2 protein expression and PGE(2) production in equine leukocytes. LPS stimulation activated p38 MAPK and increased COX-2 expression in a dose-dependent manner with maximal activation observed after 30min and 4h, respectively, at a concentration of 10 ng/ml LPS. In contrast, LPS stimulation did not affect COX-1 protein expression. Pretreatment with SB203580 or SB202190 significantly inhibited LPS-induced activation-associated p38 MAPK phosphorylation, COX-2 mRNA and protein levels, and PGE(2) production in equine leukocytes. Maximal inhibition of LPS-induced COX-2 protein expression was achieved at a concentration of 10 microM SB203580. We concluded that p38 MAPK is essential for LPS-induced COX-2 expression suggesting that p38 MAPK is a potential target for anti-inflammatory therapy during equine endotoxemia.