转基因植物中T-DNA整合的分子特征及表达

植物中不同转基因方法转化外源基因的T-DNA整合特征既具有共性,又具有特性,使得转基因的遗传在各独立转化体间呈现多样性,另外多种遗传因子和限制因素使受体植物中外源基因的表达存在下降,甚至出现基因沉默等复杂现象。本文主要对农杆菌介导及裸露DNA直接转化转基因植物中T-DNA的分子特征和转基因表达的影响因子进行了介绍和概述。转化体中转基因的遗传稳定性和表达主要取决于转基因在植物基因组中的整合位置、拷贝数及组成结构。因而,通过对具有表达水平各异的转化体进行深入的遗传分析和分子生物学研究以及转化体之间进行的比较研究,将对转基因技术自身的完善、定点整合以及更有效的利用转基因技术都具有十分重要的意义。     

转基因小鼠在现代生命科学中的应用

随着分子生物学的发展,转基因技术取得了巨大的进步,并且在现代生命科学中得到了广泛的应用.针对转基因技术在生物医学研究领域中的重要作用,结合近年来转基因技术及转基因动物的研究成果,着重介绍转基因小鼠的发展,转基因的方法及在医学中的应用,并对转基因小鼠在现代生命科学中的应用前景进行了展望.     

香蕉转基因研究进展

香蕉转基因研究逐渐成为国内外植物分子生物学研究的热点。人们从 2 0世纪 60年代开始就进行了香蕉的组织培养 ,70年代茎尖培养成功 ,近 1 0年来相继在微繁殖、单克隆抗体、分子生物学和遗传转化等方面开展了工作。香蕉转化受体系统已从茎尖发展到胚状体和愈伤组织受体系统 ,遗传转化方法也在不断改进 ,从单一的方法发展为根癌农杆菌感染法与基因枪法的结合。从香蕉组织培养、遗传转化受体系统、基因转化方法、筛选方法的研究等方面进行综述 ,并提出了一些看法。     

植物学新絮—转基因植物

一九八三年,第一株含有合成蛋白质外源基因的转基因植物成功地诞生了,它标志着植物分子生物学又进入了一个新纪元,为植物遗传工程带来了广阔的前景.之后,在各国生物学家的共同努力下,又成功地育成了许多含有各种优良外源基因的转基因植物.这种遗传工程的方法消除了传统植物育种的盲目性,突破了“亲和性”的限制,只要有理想的供体和受体DNA,遗传工程技术就能使它们的基因结合表达,并稳定地遗传给后代.大大扩大了育种的范围.     

高等植物转基因研究

1　转基因的研究意义转基因是指通过基因工程技术将外源基因导入受体细胞内的过程。自 1984年转基因烟草问世以来 ,转基因技术已经在 2 0 0多种植物中获得成功。转基因物种涉及 5 0多个 ,特别是转基因棉花、水稻、大豆、玉米、烟草、蔬菜等已在生产上发挥重大作用。利用转基因技术可以冲破物种的界限 ,实现物种间的遗传育种 ,并能改良现有物种的生物性状 ,最大限度为人类服务。不仅如此 ,转基因技术还提供了科学的研究手段 ,使人类科技实现新的飞跃成为可能。将外源基因导入受体只是转基因的第一步 ,其在受体中的命运受到一系列生理生化过程…     

中国转基因水稻的研究进展及产业化问题分析

水稻在我国粮食生产和消费中占有重要地位,也是世界上最重要的粮食作物之一。水稻转基因研究已成为当前国内外植物分子生物学和作物育种研究的热点。目前我国转基因水稻研究处于国际领先水平,有望成为转基因抗虫棉之后又一个进入产业化的转基因粮食作物,这可能将在确保我国粮食安全中发挥重要贡献。从国内外转基因水稻研发概况、我国Bt抗虫水稻生物安全评价两方面综述了我国转基因水稻产业化的前景,并在此基础上对产业化提出相关建议与对策。     

大豆转基因的研究进展

大豆转基因操作中常用的再生受体系统有：器官发生受体系统,体细胞胚胎发生受体系统,原生质体受体系统,以及所使用的转基因方法： 直菌介导法,基因枪法,PEG法等,并对今后大豆转基因存在的一些技术问题进行了探讨。     

精子介导转基因动物的研究进展

精子是高度分化的细胞,它具有潜在的结合外源DNA并在受精过程中将其转入到卵内的能力。随着受精卵的发育,外源基因将随机整合到受体基因组中,部分基因能在成体中表达,部分基因不仅能表达还能遗传给后代。一旦精子能表达外源基因,那么将很容易得到大量的转基因后代。因此,精子能携带外源基因入卵和产量丰富这两大特性使精子作为载体制备转基因动物成为一个简便的途径。精子载体法制备转基因动物无需昂贵的实验设备,亦无需专门的技术。因此,提出后就备受关注。介绍精子介导的转基因动物的制作原理及具体方法的研究进展。     

水稻转基因技术的现状及在育种上的应用

近十几年来,随着分子生物学的飞速发展,人类对植物基因的结构、功能和表达有了较为清晰的了解,分子生物学对作物育种的促进作用越来越明显。与植物基因组的研究、ＲＦＬＰ和ＰＣＲ等辅助选择手段相比较,转基因技术以其把外源基因主动导入、定向改造植物的优点日益为世人所瞩目转基因技术使基因可以在植物、动物、微生物之间相互转移,克服了物种间的隔离,已成为一种新的育种手段。     

植物转基因沉默及对策

转基因 (ｔｒａｎｓｇｅｎｅ)是指所有通过基因工程手段构建 ,导入受体生物细胞并稳定整合到该受体细胞基因组中的外源基因。人们对植物进行遗传转化的最终目的是让转基因在受体植物基因组中得到稳定整合并在当代及其子代中得到有效、稳定的表达 ,但是由于存在多种影响因子和限制因素 ,使得转基因在受体植物中的表达往往事与愿违。现实证明 ,转基因在受体植物中往往不能稳定表达 ,有时甚至完全不表达 ,出现了所谓的转基因沉默现象 (ｔｒａｎｓｇｅｎｅｓｉｌｅｎｃｉｎｇ)。转基因沉默并不等同于由于转基因在受体细胞中ＤＮＡ序列的歧变或因…     

Stable Long Term Germ-Line Transmission of Transgene Integration Sites in Mice

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.     

Transgene flow in Mexican maize revisited: Socio‐biological analysis across two contrasting farmer communities and seed management systems

The flow of transgenes into landraces and wild relatives is an important biosafety concern. The case of transgene flow into local maize varieties in Mexico (the center of origin of maize) has been intensively debated over the past 15 years, including legal, political, and environmental disputes fanned by the existence of a significant scientific controversy over the methods used for the detection of transgenes. The use of diverse approaches and a lack of harmonized methods specific to the detection and monitoring of transgenes in landraces have generated both positive and negative results regarding contamination of Mexican maize with genetically modified material over the years. In this paper, we revisit the case of transgene contamination in Mexican maize and present a novel research approach based on socio‐biological analysis of contrasting communities and seed management systems. Two communities were used to investigate how different social and biological factors can affect transgene flow and impact transgene spread in Mexico. Our results show the presence of transgenes in one community and thus support the position that transgenes are highly likely to be present in Mexican maize landraces. However, our work also demonstrates that the extent and frequency with which transgenes can be found will significantly depend on the societal characteristics and seed management systems of the local communities. Therefore, we argue that future analysis of transgene presence should include social research on the seed management practices in the sampling area so that more robust and comprehensive understandings and conclusions can be drawn.     

Generation of Chlamydomonas strains that efficiently express nuclear transgenes

The unicellular green alga Chlamydomonas reinhardtii is both an invaluable model organism for plant biology and an attractive biotechnological production system. Despite the availability of efficient methods for introduction of foreign genes into the nuclear genome of the alga, transgene expression levels are usually very poor. This is a serious limitation that has severely hampered both post-genomics research in Chlamydomonas and use of the alga in molecular farming. Here we report a solution to this problem. We have designed a genetic screen that facilitates isolation of algal strains that efficiently express introduced transgenes. The levels of accumulation of foreign protein in our expression strains are almost uniformly high in all transgenic clones and are little influenced by position effects. The possibility of expressing transgenes to high levels will greatly facilitate post-genomics research in Chlamydomonas , and will also boost exploitation of the alga as an inexpensive production host for biopharmaceuticals and other valuable compounds.     

Genetically Engineered Human Cancer Models Utilizing Mammalian Transgene Expression

Cancer models are vital to cancer biology research, and multiple cancer models are currently available that utilize either murine or human cells, each with particular strengths and weaknesses. The ability to transform primary human cells into tumors through the expression of specific transgenes offers many advantages as a cancer model, including genetic malleability and the ability to transform specific cell types. Until recently, the conversion of primary human cells into tumors through transgene expression required the use of viral genetic elements, which unfortunately adds uncertainty regarding which cancer pathways are affected and how they are affected. In recent years multiple reports have described the transformation of primary human cells into tumors using only mammalian transgenes. This review focuses on these five cancer models, comparing the different cell types which were transformed into tumors and which transgenes were expressed, as well as the cancer pathways affected in the disparate models. These genetically-engineered human cancer models offer a valuable tool to complement existing cancer models and further cancer research.     

A 'select and swap' strategy for the isolation of clones with tightly regulated transgenes.

Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression of transgenes. It is desirable that expression of such a transgene is tightly regulated, from close to zero expression in the 'off' state, to appreciable (at least physiological) expression in the 'on' state. Although there are many examples where tight regulation has been achieved, certain factors, including chromosomal position effects due to random integration of the transgene, often cause suboptimal inducibility and make the isolation of tightly regulated clones difficult and/or laborious. Here we describe a 'select and swap' strategy for the isolation, from a population of stable transfectants, of clones with tightly regulated transgenes. In this approach, a positively and negatively selectable, inducible marker gene is used to select for clones with optimal transgene regulation. After isolation of such clones, the marker gene is swapped with a linked gene of interest by the use of site-specific recombination. To test this strategy we introduced into human cells a plasmid with a tetracycline-inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site-specific recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applicability.     

Identification of anti-repressor elements that confer high and stable protein production in mammalian cells

The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number-dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.     

Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.