转基因动物的研究进展与应用前景

转基因动物指通过人为的方法将外源目的基因导入到动物染色体基因组内,使之稳定表达并能遗传给后代的一类动物.转基因动物技术是近20年来发展起来的生命科学研究领域中的一个全新技术,目前,该技术已在分子生物学、生物制药、医学及畜牧育种等方面得到应用,展现出广阔的应用前景和潜在的经济效益.笔者主要对转基因动物的研究进展、应用及其发展前景进行综述.     

转基因技术在动物营养上的应用

转基因技术的研究和应用是现代生命科学的前沿技术,已渗透到生命科学的各个领域,动物营养学的发展需要在分子水平上分析及解释营养物质对动物机体的变化调控,如生长发育、新陈代谢、遗传变异、免疫与疾病等.本文综述了转基因动物技术的方法在动物营养学中的应用.     

转基因动物研究进展

转基因动物技术是将已知的外源基因导入动物细胞并整合到基因组中,从而使其得以表达的技术。此技术将分子、细胞和个体水平统一起来,标志着基因工程已由离体操作发展到离体与载体相结合的新阶段。目前,转基因动物的制作方法主要有反转录病毒感染法、显微注射法、胚胎干细胞法和精子载体法等,每种方法都有其优缺点。转基因动物技术主要应用在人类疾病模型、生物反应器、异体器官移植和改良动物品种与性状等方面。同时转基因动物技术也存在一些技术难题和安全性问题,但发展前景被普遍看好。     

转基因动物制备方法

动物转基因技术是通过将外源性目的基因整合到动物染色体上,使其在体内整合和表达,获得表达外源基因动物的技术。该技术广泛应用于转基因动物育种、基因功能研究、药用蛋白生产和器官移植等方面。其主要方法包括逆转录病毒感染法、显微注射法、体细胞核移植法、胚胎干细胞介导法、精子介导法等。本文旨在对动物转基因的各种技术和方法进行综述,甄别各自的优缺点,为科研工作者提供一定的参考。     

转基因技术提高动物生产性能的研究进展

动物转基因技术是在基因工程、细胞工程及胚胎工程的基础上发展起来的一种综合性的生物技术.利用该技术,人类可以按照自己意愿去改变动物的遗传组成,提高动物生长率,改进动物脂肪质量、动物乳品质量以及羊毛产量和品质,还可获得用于治疗或预防人类疾病的转基因生物药品等.然而,动物转基因技术仍在探索之中,许多问题尚未解决,转基因动物的...     

动物乳腺生物反应器的研究内容及研究现状

乳腺生物反应器技术是指利用动物乳腺特异性的乳蛋白基因启动子调控元件指导外源基因在乳腺中高效定位表达,并从转基因动物乳汁中获取重组蛋白的一种转基因技术。动物乳腺是一个封闭系统,而乳腺组织是一个高效的蛋白质合成器,其合成的蛋白非常接近于天然蛋白质,具有高活性、低抗原性和高稳定性的优点,所以乳腺组织表达的药用蛋白绝大部分可以从乳腺组织中排出后利用。目前全世界从事该项商业开发的公司已有30多家,表达水平达到可     

转基因羊研究进展

转基因动物研究始于上世纪70年代,至今已在牛、羊、猪、鱼、猴、兔、狗和猫等动物身上取得转基因成功.根据目前的生命科学和生物技术的发展现状和趋势,动物转基因技术和动物克隆技术的有机结合既有迫切性又有必然性.转基因克隆动物技术在畜牧业上的应用前景也相当诱人,转基因克隆动物的研究将成为今后国际范围内生物工程领域的竞争热点.文中综述了国内外利用转基因技术培育绵羊和山羊新品种,进行羊乳腺生物反应器和生产生物材料的研究以及我国目前正在进行的转基因羊项目,在论述转基因动物发展有利方面的同时,指出了转基因羊存在的问题和今后的研究方向及发展前景.     

转基因技术在动物营养上的应用

转基因技术的研究和应用是现代生命科学的前沿技术,已渗透到生命科学的各个领域,动物营养学的发展需要在分子水平上分析及解释营养物质对动物机体的变化调控,如生长发育、新陈代谢、遗传变异、免疫与疾病等。本文综述了转基因动物技术的方法在动物营养学中的应用。     

转基因技术在动物营养上的应用

转基因技术的研究和应用是现代生命科学的前沿技术,已渗透到生命科学的各个领域,动物营养学的发展需要在分子水平上分析及解释营养物质对动物机体的变化调控,如生长发育、新陈代谢、遗传变异、免疫与疾病等。本文综述了转基因动物技术的方法在动物营养学中的应用。     

转基因猪在武汉问世

6月27日,湖北省农科院对外公布:含人类基因、其血液能取代人血提取"医用白蛋白”的"转基因猪”正式问世武汉. 湖北省农科院生物所转基因课题组经过10多年的研究,成功地在国内首次用转基因猪表达人的血清蛋白.打日,在该所的试验猪场里,记者见到3头转基因猪.华中农业大学生命科学院测定,其中蛋白最高表达量为20.3g/L,是目前国内外转基因动物肝脏表达外源蛋白的最高水平.     

农杆菌介导白三叶草高效遗传转化和转基因植株再生

利用子叶下胚轴为外植体,通过农杆菌Ti质粒介导途径,建立了白三叶草高效遗传转化及转基因植株高频率再生体系。PCR检测及Southern印迹鉴定结果表明,外源T-DNA片段已整合到转基因植株的基因组中,且多以1~2个少数拷贝存在。Northern印迹结果和对报告基因GusA编码蛋白的活性检测结果表明,目的基因在部分转基因植株中高水平表达。适宜条件下外植体遗传转化后的植株再生比例达58.23%~62.12%。与对照相比,转基因植株在外部形态上没有发生改变。     

RNAi技术在转基因猪中的应用研究进展

RNAi技术介导的转基因小鼠的出现,预示着在哺乳动物整体水平上研究靶基因的敲除成为可能。目前,针对家畜这一类较大的动物,出现了RNAi技术介导的转基因猪,为应用RNAi技术培育抗猪病毒新品种奠定基础。文章以RNAi转基因猪为代表,阐述了RNAi技术在哺乳动物细胞中特殊的现象及最有应用前景之一的抗病毒逃逸方法,并详细介绍了RNAi技术在抗病转基因猪中的研究进展。     

拟蜘蛛牵丝蛋白基因细毛羊毛囊特异表达载体的构建及其转基因细胞株的筛选

试验旨在获得具有毛囊表达特性的转蜘蛛牵丝蛋白基因细胞株。根据GenBank上发表的棒络新妇属蜘蛛的cDNA片段合成拟蜘蛛牵丝蛋白基因单体S,加倍后连入pEGFP-N1框架载体以构建真核表达载体pK-2S,转染新疆美利奴细毛羊皮肤成纤维细胞后筛选单克隆,并通过PCR在DNA、RNA水平检测阳性克隆。基因合成后测序结果显示序列正确,酶切得到正确目的条带,筛选出阳性克隆并且可以在基因组及cDNA上扩增出目的片段。本研究成功将蜘蛛牵丝蛋白基因转入新疆美利奴细毛羊细胞,并在RNA水平表达,为培育毛囊特异表达蜘蛛牵丝蛋白基因并具有更高机械性能羊毛的新型细毛羊品种奠定基础。     

褪黑素合成酶AANAT/ASMT基因过表达绵羊生物安全评价研究

旨在研究乳腺过表达褪黑素合成酶基因AANAT和ASMT（HIOMT）绵羊的生物安全性,基于本实验室前期建立的乳腺过表达褪黑素合成酶基因AANAT和ASMT（HIOMT）绵羊模型,追踪阳性转基因绵羊的生长性状数据,分析血液及尿液生理生化指标,对肠道微生物、乳中褪黑素水平及乳成分进行检测。结果表明：1）阳性转基因绵羊0、6和12月龄的体重、体长、身高和胸围4项生长指标与普通绵羊均无显著差异（P>0.05）;2）阳性绵羊血液总蛋白、白蛋白、球蛋白、胆固醇含量和各类型细胞数量以及尿液中亚硝酸盐、蛋白质和葡萄糖含量等指标均属正常,且与普通绵羊均无显著差异（P>0.05）;3）菌群测序结果表明,阳性绵羊及普通绵羊肠道微生物群落组成及优势菌群均无显著差异（P>0.05）;4）转基因绵羊血液褪黑素表达水平与普通绵羊无显著差异（P>0.05）,夜间褪黑素水平显著高于白天（P<0.05）;乳中褪黑素水平极显著高于普通绵羊（P<0.01）,乳糖含量显著高于对照羊（P<0.05）,体细胞数极显著低于普通绵羊（P<0.01）。综上所述,乳腺过表达AANAT和ASMT（HIOMT）转基因绵羊的生长发育、诸多生理生化指标、肠道微生物菌群等与对照组绵羊相比均无显著差异。此外,转基因绵羊乳中褪黑素和乳糖含量较高,体细胞数含量较低,可能是乳腺中褪黑素发挥抗炎作用,降低了乳中体细胞数。     

转抗仔猪腹泻基因α1-盐藻糖转移酶克隆猪的胰岛素样生长因子2表达分析

为探求转基因克隆猪存在早期死亡率高及生长发育异常等原因,本研究首次以转抗仔猪腹泻基因α1-盐藻糖转移酶(fucosyl transferase 1,FUT1)阳性及阴性克隆猪的脾脏、肝脏及腿肌组织为试验材料,利用qRT-PCR技术定量检测影响胎儿及仔猪早期生长发育的印记基因胰岛素样生长因子2(insulin-like growth factor 2,IGF2)的表达情况,并以普通妊娠分娩猪为对照,分析其差异及变化.结果显示,IGF2基因在3类仔猪中均存在组织表达差异性,其中肝脏组织中表达量最高,腿肌组织中表达量最低.分析发现,IGF2基因在转FUT1阳性克隆猪脾脏组织中的表达量显著高于转FUT1阴性克隆猪(P<0.05),在腿肌组织中则显著低于阴性克隆猪(P<0.05).此外,转FUT1阴性克隆猪的脾脏、肝脏组织IGF2表达量分别极显著低于、高于普通猪(P<0.01).提示IGF2基因的表达变化可能与克隆猪早期生活力弱有关,但与转FUT1基因克隆猪早期死亡率高的关系还需进一步研究.     

抗草甘膦转基因玉米AG16分子特征和抗性鉴定

抗草甘膦转基因玉米(Zea mays)能有效降低杂草防治成本,具有重要的应用前景。浙江大学转基因抗虫植物和生物安全实验室前期通过农杆菌介导法,以玉米Hi-Ⅱ品种为受体导入新型抗草甘膦基因G10evo,获得不同的抗草甘膦玉米转化系。在此基础上,筛选了具有良好草甘膦抗性的转基因玉米转化系AG16。本研究利用PCR、Western杂交、Southern杂交、ELISA等方法对AG16进行分子特征检测,并对AG16的草甘膦抗性水平进行鉴定。结果表明,G10evo在AG16中为单拷贝插入;G10evo蛋白在AG16的根、茎和叶组织中表达;ELISA分析表明,草甘膦喷洒前,嫩茎中G10evo蛋白的表达量达到9.975μg·g-1。温室中草甘膦抗性测定结果显示,AG16能抗4~8倍田间浓度的草甘膦,远高于草甘膦在实际生产中的使用量。因此,AG16草甘膦抗性水平达到生产需求,具有产业应用潜力,为培育具有自主知识产权的抗草甘膦玉米提供了种质资源。     

Ta6-SFT基因对油菜的转化及抗旱性分析

为了研究Ta6-SFT对油菜抗旱的影响,进行了Ta6-SFT对油菜纯系材料的遗传转化,获得经PCR、Southern杂交和Northern 斑点杂交验证的转基因植株。对7株T₁代转基因油菜进行了为期36 d的干旱胁迫,分析干旱胁迫36 d和复水后2 d各植株及野生型对照中的Ta6-SFT的转录水平表达和果聚糖含量,同时进行同时期丙二醛含量和相对电导率测定。结果表明,Ta6-SFT的转录水平表达与转基因植株体内的果聚糖含量、转基因植株的抗旱性呈正相关,表明Ta6-SFT在干旱胁迫下的表达增强了转基因植株的抗旱性。     

Postmortem proteolysis is reduced in transgenic mice overexpressing calpastatin

Using both in vitro and in vivo approaches, numerous studies have provided evidence that mu-calpain is responsible for postmortem proteolysis. This paper reports the effect of overexpression of calpastatin on postmortem proteolysis in transgenic mice. Transgenic mice (n = 8) with a human calpastatin gene, whose expression was driven by the human skeletal muscle actin promoter, were killed along with control nontransgenic littermates (n = 5). Hind limbs were removed and stored at 4 degrees C, and muscle samples were dissected at 0, 1, 3, and 7 d postmortem and analyzed individually. At time 0, active human calpastatin was expressed in transgenic murine skeletal muscle at a level 370-fold greater (P < 0.001) than calpastatin in control mice. Although the native isoform of this protein was degraded with storage, at 7 d postmortem, approximately 78% of at-death activity remained, indicating that degraded calpastatin retains activity. Calpain (mu- and m-) expression was unaffected (P > 0.05) by the transgene as assessed by immunoreactivity at d 0. Over 7 d, 33% of at-death 80-kDa isoform immunoreactivity of mu-calpain was lost in transgenics compared to an 87% loss in controls, indicating that autolysis of mu-calpain was slowed in transgenic mice. Desmin degradation was also inhibited (P < 0.05) in transgenics when compared to controls. Control mice lost 6, 78, and 91% of at-death native desmin at 1, 3, and 7 d postmortem, respectively; conversely, transgenic mice lost only 1, 3, and 17% at the same times. A similar trend was observed when examining the degradation of troponin-T. Interestingly, m-calpain seemed to undergo autolysis in control mice, which in postmortem tissue is indicative of proteolysis. Further investigation revealed that both mu- and m-calpain are active postmortem in normal murine skeletal muscle. In conclusion, a high level of expression of active calpastatin was achieved, which, by virtue of its inhibitory specificity, was determined to be directly responsible for a decrease in postmortem proteolysis.     

Comparison of protection levels against pseudorabies virus infection of transgenic mice expressing a soluble form of porcine nectin-1/HveC and vaccinated mice

We recently generated transgenic mice expressing a soluble form of porcine nectin-1 (PHveCIg) showing remarkable resistance to pseudorabies virus (PRV) infection. Nectin-1, also known as herpesvirus entry mediator C (HveC), is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD). In order to evaluate the level of resistance to PRV infection induced by the expression of PHveCIg in the transgenic mice, the protective effects of vaccinated and transgenic mice were directly compared. Mice were immunized with a live vaccine, through intraperitoneal injection of PRV strain Begonia (an attenuated vaccine strain deleted for gE and thymidine kinase genes) at 4 weeks before challenge. The vaccinated and transgenic mice were challenged with 10LD(50), 20LD(50) or 50LD(50) of PRV strainYS-81 via intranasal route. In the vaccinated mice, no protection was observed in the challenges with 20LD(50) and 50LD(50). Only two out of six vaccinated mice survived in the challenge with 10LD(50). In contrast, four transgenic mouse lines showed significant resistance to PRV infection, although the survival rates varied in the challenge with each viral dose. These results demonstrate clearly the high potential of transgenic strategy in control of pseudorabies.     

Supplements of transgenic malt or grain containing (1,3-1,4)-beta-glucanase increase the nutritive value of barley-based broiler diets to that of maize

1. A diet with addition to normal barley of malt from transgenic barley expressing a protein engineered, thermotolerant Bacillus (1,3-1,4)-beta-glucanase during germination has previously been demonstrated to provide a broiler chicken weight gain comparable to maize diets. It also reduced dramatically the number of birds with adhering sticky droppings, but did not entirely eliminate sticky droppings. One of the objectives of the broiler chicken trials reported here was to determine if higher concentrations of transgenic malt could alleviate the sticky droppings. 2. Another aim was to investigate the feasibility of using mature transgenic grain containing the thermotolerant (1,3-1,4)-beta-glucanase as feed addition and to compare diets containing transgenic grain to a diet with the recommended amount of a commercial beta-glucanase-based product. 3. Inclusion of 75 or 151 g/kg transgenic malt containing 4.7 or 98 mg/kg thermotolerant (1,3-1,4)-beta-glucanase with 545 or 469 g/kg non-transgenic barley instead of maize yielded a weight gain in Cornish Cross broiler chickens indistinguishable from presently used maize diets. The gene encoding the enzyme is expressed in the aleurone with a barley alpha-amylase gene promoter and the enzyme is synthesised with a signal peptide for secretion into the endosperm of the malting grain. 4. Equal weight gain was achieved, when the feed included 39 g/kg transgenic barley grain [containing 66 mg/kg thermotolerant (1,3-1,4)-beta-glucanase] and 581 g/kg non-transgenic barley instead of maize. In this case, the gene encoding the enzyme has been expressed with the D-hordein gene (Hor3-1) promoter during grain maturation. The enzyme is synthesised as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeted into the storage vacuoles. Deposition of the enzyme in the prolamin storage protein bodies of the endosperm protects it from degradation during the programmed cell death of the endosperm in the final stages of grain maturation and provides extraordinary heat stability. The large amount of highly active (1,3-1,4)-beta-glucanase in the mature grain allowed the reduction of the transgenic grain ingredient to 0.2 g/kg diet, thus making the ingredient comparable to that of the trace minerals added to standard diets. 5. A direct comparison using transgenic grain supplement at the level of 1 g/kg of feed with the standard recommended addition of the commercial enzyme preparation Avizyme 1100 at 1 g/kg yielded equal weight gain, feed consumption and feed efficiency in birds fed a barley-based diet. 6. The production of sticky droppings characteristic of broilers fed on barley diets was avoided with all 9 experimental diets and reduced to the level observed with a standard maize diet by supplementation with transgenic barley. 7. The excellent growth and normal survival of the 400 broilers tested on barley diets supplemented with transgenic grain or malt showed the grain and malt not to be toxic. 8. The barley feed with added transgenic grain or malt containing thermotolerant (1,3-1,4)-beta-glucanase provides an environmentally friendly alternative to enzyme additives, as it uses photosynthetic energy for production of the enzyme in the grain and thus avoids use of non-renewable energy for fermentation. The deposition of the enzyme in the protein bodies of the grain in the field makes coating procedures for stabilisation of enzyme activity superfluous. 9. Barley feed with the small amount of transgenic grain as additive to normal barley provides an alternative for broiler feed in areas where grain maize cannot be grown for climatic reasons or because of unsuitable soil and thus has to be imported.