外源基因在转基因动物乳腺中的特异性表达研究

动物基因工程研究最大的突破就是转基因动物研究的进展,自八十年代初第一批转基因小鼠问世以来,转基因动物的研究已从方法学研究步入了应用性研究阶段,转基因动物除作为研究工具广泛应用于发育生物学、免疫学、遗传学以及医学等生命科学领域外,外源基因在转基因动物的特异性表达,尤其是在乳腺的表达,又可将转基因用作生物反应器进行了生物活性蛋白的生产而于商业生产,在转基因动物乳腺的特异性表达研究中,寻找乳蛋白基因调控     

欧美爆发转基因食品战

从本世纪80年代世界上第一例转基因植物的诞生,到目前全世界约有2780万公顷的转基因作物,到诞生转基因动物生物反应器大量的生产医用蛋白,人类将最新的生物技术应用于医药、食品、农业领域,以摆脱自然对传统作业的限制。 转基因植物是把来源于任何生物甚至人工合成的基因转入植物,这可能是自然界无法发生的,人们也无法预测基因进入一个新的遗传背景中会产生什么样的作用。转基因动物,一是将正常人的基因片断导入动物体内,让这种基因在哺乳动物体内表达,并通过该动物分泌的奶或其他组织,提取获得具有活性的分泌物质,获得大量廉价的珍稀药物;另一方向是利用转基因动物培养人体器官,     

转基因动物乳腺生物反应器研究

以转基因动物的乳腺组织即乳腺生物反应器生产药用蛋白 ,是近年来生物技术领域发展的重要方向 .利用转基因动物的乳腺组织能够生产出具有完全生物活性的药用蛋白 ,纯化简单 ,投资少 ,成本低 ,对环境没有任何污染 ,可以获得巨额经济效益 .人凝血因子Ⅸ是治疗血友病Ｂ的特异药用蛋白 ,我们和上海市医学遗传学研究所合作 ,在上海市科委资助下 ,开展了ｈＦＩＸ在转基因动物乳腺中特异表达研究 ,构建乳腺特异表达的ｈＦＩＸ载体和表达系统 ,通过显微注射制备乳腺特异表达ＦＩＸ的转基因小鼠和羊 ,在转基因奶山羊中检测到人凝血因子Ⅸ的表达 ;促血…     

转基因植物药物的开发研究评述

基因工程的完善与发展为利用转基因植物作为生物反应器来开发与生物转基因植物药物提供了可靠的技术基础，目前已利用转基因植物作为反应器生产出了多种转基因植物药物，其中包括多种药品和疫苗，已有的转基因植物药物的临床实验及国内外的应用表明转基因植物药物安全是可靠的，随着表达效率和遗传稳定性的不断提高，转基因植物药物的开发研究将会在21世纪得到更大的发展。     

人肝细胞再生增强因子双顺反子表达载体的构建及表达

PCR扩增人肝细胞再生增强因子(Human augmenter of liver regeneration,ALR)基因,将其插入质粒pIRES2-EGFP多克隆位点中,构建成新霉素(Neo)、增强绿色荧光蛋白(Enhanced green fluorescence protein,EGFP)双标记基因且EGFP和ALR基因为双顺反子的真核表达载体pAIE.另外体外培养绵羊胎儿成纤维细胞(Sheep fetal fibroblast cells,sFFCs),脂质体lipofectAMINETM介导pAIE转染sFFCs,经过筛选后形成单克隆细胞,RT-PCR和免疫组化方法进一步检测ALR基因的表达.进一步将转基因细胞饥饿培养,体外成熟培养绵羊卵母细胞146个,进行体细胞核移植,经融合、激活后,在培养液中进行发育培养,对发育到8细胞胚胎在激光共聚焦显微镜下观察;同时利用免疫组化检测ALR基因在胚胎中的表达.结果表明:由IRES连接的EGFP和ALR基因在sFFCs内同时存在并表达;得到45个8细胞体细胞克隆胚胎,其中14个发育形成囊胚,EGFP和ALR基因在体细胞克隆胚胎中同时存在并表达.因此由IRES连接标记基因和目的基因,以标记基因指示目的基因的表达,可减少检测目的基因的繁琐手段,可得到高纯度表达ALR基因的转基因细胞和胚胎,为生产药用蛋白治疗肝脏疾病奠定实验基础.     

家养动物的遗传工程:来自鼠遗传模型报告

本文对从转基因鼠所得到的大量观察进行了报道,以便检验将基因直接导入家养动物的可行性。外源基因的组织特异的或一般的表达已经实现,并且有证据表明它可与内源基因相配合进行活动,这些结果表明参与某些有动物技术价值的特征行为(如食物消化,泌乳,免疫反应)的器官(如肝、乳腺)或组织(如外分泌腺、B淋巴细胞)的活动,通过基因转移得到适当的促进。目前,偶而也能观察到外源基因的异常表达或传递;有时转基因动物的生殖力会严重下降。这些失控行为及至今尚在使用的将基因导人生殖系的低效率方法,对于基因工程在家养动物中的应用仍是一个主要的障碍。     

转基因小鼠与肿瘤研究

转基因小鼠的出现有力地推动了肿瘤研究的发展。具有癌症倾向的转基因小鼠,是从DNA水平开展有关人类恶性肿瘤发生、生长、转移和防治研究的良好活休模型。肿瘤形成与控制细胞生长和分化的多种基因特别是癌基因、抑癌基因的遗传改变有关。在利用转基因小鼠进行实验研究时,对由相同结构基因建立的不同转基因小鼠家系在癌基因表达上的时空差异性,应当给予足够的重视。此外,实现转基因动物生产的专业化、产业化,将会加速我国生命科学领域中转基因动物应用研究的进程。     

家蚕生物反应器与家蚕实验动物化饲育技术

以家蚕为生物反应器 (重组家蚕核型多角体病毒表达系统 )生产基因工程产品 ,具有成本低、安全、产品产量与活性高的优点。随着近年生命科学和生物技术的迅猛发展 ,需要发展新的实验动物品种、特别是利用低等动物代替哺乳动物 ,大力开发动物资源型功能 ,造福人类。本文报道我所利用家蚕表达外源基因与家蚕实验动物化饲育技术方面一些研究成果。由家蚕细胞、转移载体、修饰的BmNPV、家蚕幼虫或蛹组成家蚕表达系统。获得了含有日本血吸虫脂肪酸结合蛋白基因的重组病毒 (rsj14NPV)和含有日本血吸虫 2 8KDGST基因的重组病毒 (rsj2 8NPV)。r…     

绵羊乳腺特异表达hALR基因载体的构建

以绵羊β-乳球蛋白(β-lactoglobulin，BLG)基因5’端0．8kb片段，作为目的基因人肝再生增强因子(Human augmenter of liver regeneration，hALR)乳腺特异表达启动子，人巨细胞病毒(Human cytomegalovirus，CMV)启动子为绿色荧光蛋白基因(Enhanced Green fluorescence protein，EGFP)的启动子，构建了ALR基因乳腺特异表达而EGFP基因非组织特异性真核表达载体．这将为利用乳腺生物反应器生产人肝再生增强因子转基因动物及转基因早期胚胎鉴定奠定基础。     

转基因动物检测方法的比较研究

用兔乳清酸性蛋白基因启动子及其远端上游区、免乳清酸性蛋白基因终止子及人瘦蛋白基因cDNA经一系列的亚克隆(Subcloning)构建了用于在转基因动物乳腺中表达人瘦蛋白的特异性表达载体，并采用显微注射的方法制作转基因小鼠动物模型。本文报道在检测转基因小鼠时出现的问题及其解决方案。     

Application of a nuclear localization signal gene in transgene mice

Efficient gene transfer by cytoplasm co-injection will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine α-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.     

Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice

Milk contains a large amount of protein, most of which consists of a few major species synthesized in the mammary gland. The genes encoding these proteins are single-copy, and expressed during pregnancy and lactation. Although beta-lactoglobulin (BLG) is the major protein in the whey of ruminants, it is not present in rodent milk. We have generated transgenic mice carrying the sheep BLG gene, and show that in such mice, BLG is specifically and abundantly expressed in the mammary gland during lactation. This results in a remarkable alteration of milk composition. These findings suggest that the manipulation of milk composition by gene transfer has considerable potential for the improvement of dairy animals.     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.     

Correction of RNA aberrant splice increases foreign gene expression in transgenic mice

Transgenic mice with mammary gland secreting human granulocyte colony stimulating factor (G-CSF) were produced using mice whey acid protein gene promoter. It was found that there was very low expression level in mammary gland. Human G-CSF cDNA was obtained by RT-PCR from transgenic mice mammary gland. Sequence analysis showed that this G-CSF gene deleted the 4th exon, and compared with human G-CSF genomic DNA, there were donor and acceptor splice sites in the deletion fragment. It was considered that the 3rd and 4th introns also delete in G-CSF fragment. The transgenic construct was corrected by deleting the 3rd and 4th introns to construct the minigene, which was used to produce transgenic mice by microinjection. Northern blot showed that G-CSF expression using the new construct increased 5.4 times as that before in transgenic mice. The results suggested that it was possible that RNA aberrant splice result in low expression in transgenic mice.     

四川省八角莲属药用植物资源

八角莲属(Dysosma R.E.Woodson)为我国小科植物所特有的属,共7种。四川省为主产区,有6个种。本文对四川省的6种八角莲属植物的产地、生境、主要化学成份、药用部位及功效进行了整理。绘制了四川省本属植物水平分布图,编写了分种检索表。     

生物技术在海洋鱼类苗种培育中的应用:现状与前景

围绕海洋鱼类苗种培育过程中饵料和病害这两个关键问题,就基因克隆、重组蛋白、DNA疫苗、抗菌肽和转基因技术等生物技术和产品在鱼类中的研究现状和前景进行讨论和分析,提出应针对鱼苗的营养需求和消化吸收的生理特点,有效利用基因工程产品;同时,要研究和采用新的病害控制技术,提高苗种成活率.苗种培育是海洋鱼类养殖业发展的基础,在苗种培育中加强生物技术的应用,将大力推动海洋鱼类养殖业的健康可持续发展.     

基因枪法在植物转基因中的应用

基因枪法是是指用鸽粉或金粉包裹外源DNA,而后依靠基因枪装置,利用高压氦气冲击波加速微弹去穿透植物细胞壁和细胞膜,使外源DNA进入植物细胞并整合到植物细胞染色体组中,从而达到稳定遗传和表达的目的。它多用于对农杆菌不敏感的物种和基因型,用外源DNA包被的钨粒对大豆茎尖分生组织进行轰击,获得大豆转基因植株,此后,这一技术便很快在大豆中得到了应用。虽然它有缺点,但可与其他转基因技术结合使用。农杆菌介导法和基因枪轰击法各有自己的优缺点,它们之间并不是相斥的M相反,通过结合两种方法的特性,互相取长补短,就有可能提高转化效率。     

米根霉发酵生产L( )-乳酸研究进展

综述了米根霉发酵生产L( )-乳酸的研究进展.米根霉是生产L( )-乳酸的理想菌种,目前主要集中在菌株的选育、发酵工艺的优化和提取分离操作以及新型反应器的设计等方面的研究,通过控制米根霉菌体的形态可提高菌株产酸的能力,操作简便.提出今后应从利用基因工程等技术定向选育出高产L( )-乳酸的基因工程菌株,优化发酵工艺,改进发酵设备和选择合适的固定化载体等方面进一步研究,从而降低生产L( )-乳酸的成本,加大乳酸衍生物产品的开发与利用,扩大L( )-乳酸的应用领域.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.)

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutin1 gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors (pUUOAH). The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of Cry1Ah protein in the construct containing the ubi1 intron (pUUOAH) was 20% higher than that of the intronless construct (pUOAH). Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubi1 intron was higher than that of the intronless construct. These results indicated that the maize ubi1 intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently