转基因动物中基因转移和表达的分子机制

本综述着重讨论在转基因动物中外源基因转移及表达的分子机制,包括外源基因在宿主染色体上整合的分子基础及其影响因素;启动子、内含子、增强子、反式作用因子及载体等各种因素对外源基因在宿主内表达的调节。     

两种不同的转基因细胞构建体系的比较

目的：比较目的基因在两种不同表达体系构建的转基因细胞上的表达，选择更好的构建体系。方法：脂质体法将重组真核细胞表达载体pcDNA3／PD-L1导入L920细胞，G418(600μg/ml)筛选抗性细胞株，同时将重组逆转录病毒载体pGEZ-Term／PD-L1与辅助载体共转染293T细胞，包装病毒颗粒，其上清感染L929细胞后，经Zeocin(500μg/ml)筛选抗性细胞。流式细胞仪(FCM)检测目的蛋白在两种细胞膜上的表达。结果：两种体系对目的基因的瞬时表达无明显差别；用pcDXA3构建的转基因细胞膜上目的分子表达稳定性差，表达率低，而用pGEZ-Term构建的则目的蛋白稳定高表达，表达率近100％。结论：两种构建体系均能瞬时表达目的分子，pGEZ-Term表达体系更适于构建长期稳定表达目的基因的转基因细胞。     

转基因小鼠的插入突变与基因克隆——方法,成就与展望

转基因技术成为研究基因的四维时空中的表达特性和功能的一种重要手段，在新基因的功能鉴定及人类疾病模型的建立中得到了重要的应用。由于转基因插入内源基因组往往引起内源基因的插入突变，这种突变占转基因小鼠“产品”的５￣１０％，比自发突变率要高得多；更为重要的是，利用已知ＤＮＡ序列的转基因作为探讨可以对具有一定突变表型的、发生插入突变的内源基因进行染色体定位和分离，从而开辟了一条利用外源基因的基因诱陷作用而     

人源轮状病毒vp7基因的克隆与转基因植物研究

目的：克隆人源轮状病毒外壳蛋白vp7基因，以制备转基因植物疫苗。方法：用RT-PCR方法制备vp7基因，以植物高效表达质粒PBI121为载体，构建重组DNA质粒。重组体用载体上的通用引物为测序引物，鉴定克隆的正确性。再将鉴定过的重组质粒用农杆菌介导法转染马铃薯外植体，分别用PCR、Western blot法鉴定阳性转化植株中vp7基因的表达。结果：成功地将vp7转入马铃薯植株中，并且在转化植株中检测出了vp7的表达。结论：成功地构建了重组PBI121/hvp7质粒，获得表达外源基因vp7的转基因马铃薯。     

病毒研究的新体系——转基因动物

转基因动物是通过遗传操作，使外源基因在体内稳定遗传的动物。转基因动物体系是研究基因功能的新手段，在病毒学研究中有广泛的运用前景。越来越多的含有病毒基因、病毒受体基因的转基因小鼠用于病毒与受体，病毒与宿主相互作用和功能的研究，从而探讨病毒的致病机理及防治方法。本文就近年来之方面有关问题的现况进行综述。     

液压转基因技术

 外源基因靶向进入动物体内并高效表达是研究基因功能和进行基因治疗的基础。近几年建立的液压转基因技术能简便、快速、安全、高效地将质粒靶向导入动物体内，并能在靶部位高效、稳定表达。本文介绍了液压转基因技术概念、机理及其应用等。     

优化反向PCR法对hTF转基因小鼠整合位点旁侧序列分析

自1982年世界上首次通过转基因动物技术获得＂巨鼠＂[1]以来,转基因动物的研究发展迅速。随着多种转基因动物的成功获得,研究者设想利用动物表达外源基因,以动物个体作为一个反应器来生产有价值的蛋白质。     

时空调控 miR-195敲减转基因载体的构建及验证

目的：构建时空调控 miR-195敲减转基因载体并验证其活性。方法通过分子克隆技术构建含有神经特异性启动子、四环素诱导表达系统、miR-195海绵、绿色荧光蛋白及绝缘子等调控元件的转基因载体并测序鉴定,利用细胞转染技术将该载体瞬时转染入 SH-SY 5 Y 细胞,经荧光显微镜观察其荧光表达,利用实时定量 PCR 技术及蛋白免疫印迹检测miR-195含量及 GFP 蛋白表达。结果通过将 Tet-on 四环素诱导系统中反义转录激活子 rt-TA 克隆到 NSE 启动子下游,以及 TRE-Tight 的多克隆位点内引入 miR-195海绵携带 IRES 介导的 EGFP ,获得双质粒 Tet-on 表达载体。以 pcDNA 3.1＋质粒为骨架,3个不同区域的双拷贝 cHs 4绝缘子核心片段为间隔,将其调控元件（ NSE-rtTA ）和反应元件（ TRE-miR-195 sponge-IRES-EGFP ）串联成单一转基因载体。该载体经测序验证序列正确,瞬时转染入 SH-SY 5 Y 细胞,经强力霉素诱导36 h 后细胞内可见明显绿色荧光蛋白表达,且 miR-195表达水平明显降低。结论成功构建神经系统特异性 miR-195敲减转基因载体,并具有时空调控的特点,为进一步建立转基因动物模型奠定基础。     

慢病毒载体法制备荧光素酶转基因小鼠

目的基于慢病毒介导的转基因方法制备荧光素酶（Luc）转基因小鼠。方法制备携带Luc基因的慢病毒,将其注入小鼠单细胞受精卵卵周隙以感染受精卵,然后将胚胎移植进假孕母鼠体内以获得仔鼠,应用小动物活体成像仪及PCR等在蛋白和DNA水平上筛选和鉴定Luc转基因小鼠。结果移植慢病毒隙感染后的成活胚胎63枚。将其移植至3只假孕母鼠,其中2只怀孕,共生仔鼠11只;利用小动物活体成像仪检测Luc表达,在蛋白水平证实11只F0代中,3只（命名为S1、S2、S3）表达Luc;DNA水平检测证实,3只Luc阳性小鼠的基因组中整合有外源转基因Luc。此外,Luc转基因首建鼠基因组中整合的Luc转基因可稳定遗传至下一代,并能正常表达。Luc转基因小鼠主要脏器如睾丸、肾脏、胃、肠、肺、脑、胸腺、肝脏和心脏等均可见Luc信号,但不同脏器间Luc强度有差异。结论成功制备Luc报告基因转基因小鼠。     

乳腺直接注射质粒DNA的转基因暂时性表达研究进展

本综述了对动物乳腺直接注射质粒ＤＮＡ进行验证转基因动物乳腺表达载体正确性的进展，对所注射的质粒ＤＮＡ的制备方法，浓度及其动物乳腺表达的可能机制进行了讨论。     

Immunologic basis of vaccine vectors

Efforts to make vaccines against infectious diseases as well as immunotherapies for cancer, autoimmune diseases and allergy have utilized a variety of heterologous expression systems, including viral and bacterial vectors, as well as DNA and RNA constructs. This review explores the immunologic rationale and provides an update of insights obtained from preclinical and clinical studies of such vaccines.     

Ectopic expression of a cecropin transgene in the human malaria vector mosquito Anopheles gambiae (Diptera: Culicidae): effects on susceptibility to Plasmodium

Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication efforts. Manipulating the endogenous immune response of mosquitoes such as the temporal and special expression of antimicrobial peptides like cecropin may result in a refractory phenotype. Using transgenic technology a unique pattern of expression of cecropin A (cecA) in Anopheles gambiae was created such that cecA was expressed beginning 24 h after a blood meal in the posterior midgut. Two independent lines of transgenic An. gambiae were created using a piggyBac gene vector containing the An. gambiae cecA cDNA under the regulatory control of the Aedes aegypti carboxypeptidase promoter. Infection with Plasmodium berghei resulted in a 60% reduction in the number of oocysts in transgenic mosquitoes compared with nontransgenic mosquitoes. Manipulating the innate immune system of mosquitoes can negatively affect their capacity to serve as hosts for the development of disease-causing microbes.     

Transgenic animal models: new avenues in cardiovascular physiology

 Application of molecular genetic tools to inherited cardiovascular disorders has provided important insights into the molecular mechanisms underlying cardiomyopathies, arrhythmias, blood pressure regulation, and atherosclerosis. In addition, alteration of gene expression has been observed under common cardiovascular conditions such as cardiac hypertrophy and heart failure. Recent advances in transgenic and gene-targeting approaches allow a sophisticated manipulation of the mouse genome by gene addition, gene deletion, or gene modifications. These transgenic models enable the dissection of in vivo pathways responsible for these complex disease phenotypes. This review describes tissue-specific promoters suitable for targeting candidate genes to the cardiovascular system as well as a number of valuable transgenic animal models of blood pressure regulation, atherogenesis, defects in the coagulation system, cardiac hypertrophy, myocarditis, cardiomyopathies, and heart failure. Limitations and difficulties associated with these transgenic approaches are discussed. Animal models which may provide a basis for future gene therapy of cardiovascular diseases are introduced. Finally, methods are described to regulate the spatial and temporal expression level of a transgene, to inactivate a target gene in a tissue-specific manner, and to introduce specific mutations into the genome. These recent advances in transgenic technology are expected to have a considerable impact on cardiovascular research in the near future. Received: 1 February 1996 / Accepted: 13 August 1996     

利用转基因植物表达肿瘤治疗相关蛋白

植物基因工程的诞生使得植物成为了一个高效的宿主生物反应器,可用来生产各种异源蛋白.近年来,利用植物生产各种药用蛋白的研究越来越引人注目,比如用于治疗肿瘤及癌症的蛋白.传统的药用蛋白生产方式成本高、产量小,远远不能满足人们的需要.利用植物基因工程方法培育的转基因植物为解决这一问题带来了希望.本文综述了一些与癌症及肿瘤治疗作用相关的蛋白在植物体内表达情况的研究进展.     

Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies

Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions. The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4(+) T cells develop normally. Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2. CD11c(+) dendritic cells express high levels of DR and DQ. Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.     

四环素调控的小鼠LAIR-1/CD305转基因小鼠的初步建立

目的：建立四环素调控的小鼠LAIR-1/CD305转基因小鼠,为进一步研究mLAIR-1分子的体内功能奠定基础.方法：构建pBI-5-mLAIR-1载体,显微注入B6D1F1受精卵,PCR检测新生小鼠基因组DNA中LAIR-1与荧光素酶（Luciferase）基因.将mLAIR-1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含盐酸强力霉素（Dox）的培养基进行培养,检测细胞裂解液中荧光素酶活性.将荧光素酶表达依赖Dox小鼠与C57BL/6交配,采用PCR对子代鼠进行检测.结果：共获得9只首建鼠,其目的基因表达高度依赖Dox,并得到其中5只首建鼠的F1代小鼠.结论：获得了四环素调控的小鼠LAIR-1转基因小鼠,可用于该分子体内功能的研究.     

外源基因在巴斯德毕赤酵母中的表达

酵母是单细胞真核生物 ,既具有类似原核生物的生长特性 ,又具有一般真核生物的细胞生物学特性。巴斯德毕赤酵母是新近发展起来的新型表达系统 ,许多有应用价值的外源基因成功地在其中表达 ,使其日益受到关注。本文就毕赤酵母的生物学特性、表达系统的构建和分泌蛋白的翻译后修饰等方面进行综述     

Herpesvirus saimiri-based gene delivery vectors

Herpesviruses possess a number of characteristics which make them promising gene delivery vectors. These include their capacity to package large amounts of heterologous DNA and an ability to establish persistent, lifelong infections, where the viral genome remains as a circular non-integrated episome. Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus and is currently being developed as a potential gene delivery vector. In addition to the above properties, HVS-based vectors have the ability to infect a wide range of human cell lines and primary cultures with high efficiencies. Moreover, upon infection the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny cells upon division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained heterologous gene expression. As such, it offers the characteristics of an artificial chromosome combined with a highly efficient delivery system. This review aims to describe the assessment of HVS-based vectors in both in vitro and in vivo studies, highlighting new developments and possible applications for the treatment of genetic diseases.     

Ubiquitous expression of human SCA2 gene under the regulation of the SCA2 self promoter cause specific Purkinje cell degeneration in transgenic mice

The objective of this work was the generation of an animal model of the SCA2 disease for future studies on the benefits of therapeutic molecules and neuropathological mechanisms that underline this human disorder. The transgenic fragment was microinjected into pronuclei of B6D2F1 X OF1 mouse hybrid strain. For Northern blots, RNAs were hybridized with a human cDNA fragment from the SCA2 gene and a mouse beta-actin cDNA fragment. Monoclonal antibody directed to the N-terminal of the ataxin 2 protein with 22Q was used for Western blot analysis. A rotating rod apparatus was utilized to measure motor coordination of mice. Immunohistochemical detection of Purkinje neurons was performed with anti-calbindin 28K as primary antibody. Ubiquitous expression of the SCA2 transgene with 75 CAG repeats regulated by the SCA2 self promoter was obtained after generation of our transgenic mice. Analysis of transgenic mice revealed significant differences of motor coordination compared with the wild type littermates. Specific degeneration of Purkinje neurons and transgene over-expression in the brain, liver and skeletal muscle, rather than in lungs and kidneys was also observed, resembling the expression pattern of the ataxin 2 in humans.     

CCL27-transgenic mice show enhanced contact hypersensitivity to Th2, but not Th1 stimuli

CCL27 is one of the CC chemokines produced by epidermal keratinocytes and is suggested to be involved in the pathogenesis of inflammatory skin diseases. To clarify the contribution of CCL27 in skin inflammation, we created transgenic C57BL/6 mice that constitutively produce CCL27 in epidermal keratinocytes. These mice had high serum CCL27 levels and did not show any phenotypical change. Thus we stimulated these mice with various reagents by single and repeated application. Interestingly, only contact hypersensitivity to repeated application with fluorescein isothiocyanate was significantly enhanced in transgenic mice compared to non-transgenic mice. Under this condition, the numbers of inflammatory cells, CCR10-positive cells, CCR4-positive cells and cutaneous lymphocyte-associated antigen-positive cells were increased, and IL-4 mRNA expression was higher in the lesional skin of transgenic mice. Increased number of mast cells and higher serum IgE levels, which were similar to atopic dermatitis, were also observed. These results indicated that CCL27 modified inflammation by attracting CCR10-positive and CCR4-positive cells into the lesional skin, and may participate in the pathogenesis of Th2-shifted skin diseases such as atopic dermatitis.