两种不同终止子在转基因鲤鱼中的促生长效应

转基因构建体中启动子的选择会直接影响转植基因的活性, 近年来有研究表明转基因构建体中终止子的选择会一定程度地影响转植基因的活性。为了更好地筛选转基因构建体和培育快速生长的转“全鱼”生长激素(Growth hormone, GH)基因鱼, 文章用鲤鱼β-actin基因终止子和生长激素基因终止子分别构建了转基因构建体, 显微注射得到转“全鱼”GH基因鱼P0代养殖群体, 比较两种不同终止子构建体的活性。统计分析发现, 生长激素基因终止子构建体的养殖群体的体重频率呈正态分布且平均体重显著高于β-actin基因终止子构建体的养殖群体, β-actin基因终止子构建体的养殖群体的体重频率呈右倾趋势的非正态分布。值得一提的是在混合养殖组中得到一条生长最为快速的鲤鱼证实为转基因阳性且为生长激素基因终止子构建体的转基因鲤鱼。该结果表明转“全鱼”生长激素基因鲤鱼可快速生长, 并能将转植基因向下代遗传。实验结果提示生长激素基因终止子构建体比β-actin基因终止子构建体表现的促生长活性要强。     

快速生长转基因鲤鱼可望在中国实现商品化

由中国科学院院士、中国科学院水生生物所朱作言研究员主持的“快速生长转基因鲤鱼的中试研究”已通过成果鉴定。中国科学院李振声院士、中国工程院刘筠院士和林浩然院士等专家鉴定认为,该项研究建立了快速生长转基因鲤鱼的高效、安全养殖模式;对转“全鱼”生长激素基因鱼食品消费安全进行了严密的科学实验,证实了转“全鱼”生长激素基因鱼的食品消费安全性,为转“全鱼”生长激素基因鲤鱼的大规模商品化生产提供了科学依据,研究成果居国际领先水平。 朱作言院士主持的该项研究,筛选获得了快速生长的转“全鱼”生长激素基因黄河鲤鱼核心群200尾,其生长速度比对照鱼快140％以上。试验证明：转基因鱼怀卵量略低于普通黄河鲤鱼,受精率和孵化率与对照鱼无显著差异,转“全鱼”生长激素基因黄河鲤鱼具备大规模苗种繁育生产能力;转“全鱼”生长激素基因鲤鱼F1代的大规模养殖不仅增产,而且可降低养殖成本,并为转基因鱼的进一步选育提供材料;转“全鱼”生长激素基因三倍体鱼平均体重增长速度比对照鱼提高15％,铒料利用效率提高11．1％,由此建立了转“全鱼”生长激素基因高效、安全的养殖模式。试验还证明：摄食转“全鱼”生长激素基因鱼对小鼠的生长、脏器发育、血液生理生化指标、繁殖能力及其后代的生长发育均无影响。为此,专家建议,尽快推广养殖转基因三倍体鲤鱼,在我国建立世界首例转基因动物品种商品化生产的范例。杨淑培     

外源生长激素基因在蓝太阳鱼中的整合、表达和遗传

通过基因重组, 将石斑鱼生长激素基因编码序列克隆到鲤鱼βactin基因启动子下游, 构建了“全鱼”生长激素基因表达载体pCAecGHc。采用显微注射法, 研制出转“全鱼”生长激素基因蓝太阳鱼。经过PCR、PCR Southern杂交、RT PCR等技术对转植基因在转基因蓝太阳鱼中的整合和表达情况进行了检测。结果表明:转植基因在两批次P0 转基因蓝太阳鱼中的整合率为5 .60%和12. 26%, 并在转基因鱼中得到了正确表达, 表现为嵌合性表达。对转基因蓝太阳实验鱼进行了对照养殖实验, 初步显示转基因蓝太阳鱼具有较快的生长表型效应, 比对照组生长速度快20%-40%左右。应用近交策略培育出了两个转基因蓝太阳鱼F1 品系, 检测表明, 转植基因在两个品系的整合率分别为22 .03%和40 .8%。结果表明: 转植基因通过性腺传递给了子代, 同时也证实转基因P0 代的生殖腺为转植基因的嵌合体。     

鱼类基因转移育种的几个问题

１９８５年,世界上第一批转基因鱼诞生。随后,鱼类基因转移迅速应用到培育高产、优质和抗逆的养殖鱼类新品种,并在解决发育生物学分子生物学和生理学等方面的难题中发挥了重要作用。目前,研究者已经建立了三种成熟的鱼类基因转移方法,即显微注射、电脉冲和精子携带法,证实了转移大受体鱼基因组中的整合、性腺传递、表达和转译表达产物的生物活性。最近,“全鱼”生长激素（ＧＨ）基因的克隆与应用,使快速生长转ＧＨ基因鱼的研     

国内信息

快速生长转基因鲤鱼可望在中国实现商品化由中国科学院院士、中国科学院水生生物所朱作言研究员主持的“快速生长转基因鲤鱼的中试研究”已通过成果鉴定。中国科学院李振声院士、中国工程院刘筠院士和林浩然院士等专家鉴定认为 ,该项研究建立了快速生长转基因鲤鱼的高效、安全养殖模式 ;对转“全鱼”生长激素基因鱼食品消费安全进行了严密的科学实验 ,证实了转“全鱼”生长激素基因鱼的食品消费安全性 ,为转“全鱼”生长激素基因鲤鱼的大规模商品化生产提供了科学依据 ,研究成果居国际领先水平。朱作言院士主持的该项研究 ,筛选获得了快速生…     

转基因鱼生态风险评价及其对策研究进展

遗传改良的转基因鱼具有许多优良经济性状, 但转基因鱼迄今尚未进行商业化养殖, 主要原因之一在于对转基因鱼逃逸或放流到自然水体中可能产生的生态风险的担忧. 本文以具有快速生长特性的转生长激素(GH)基因鱼为对象, 分析了转基因鱼潜在生态风险的实质, 简要综述了通过单因子表型与适合度分析、数学模型推演研究转基因鱼生态风险的现状, 阐述了利用人工模拟生态系统开展转基因鱼生态风险评价的新思路及最新研究成果, 同时评述了采用三倍体途径控制转基因鱼生态风险的策略及原理; 在此基础上, 提出生态风险评价与生态风险防范策略是转基因鱼育种研究体系中不可或缺的重要组成、必须与育种研究同步进行的观点, 以期为转基因鱼育种及生态风险评价和对策研究提供启示.     

转青鱼生长激素基因异源四倍体鲫鲤

生态安全性是转基因鱼走向市场的瓶颈,通过转基因四倍体鱼同转基因二倍体鱼杂交获得不育的转基因三倍体鱼是解决该问题的有效途径之一.本研究构建了青鱼β-actin基因启动子和青鱼生长激素（GH）基因精确连接的＂全鱼＂基因pbcAbcGHc;并采用显微注射法将pbcAbcGHc导入异源四倍体鲫鲤受精卵.对照养殖结果表明,150日龄的转基因异源四倍体鲫鲤原代（P0）的体重及体长明显大于对照组.选择60尾P0代转基因异源四倍体鲫鲤,采用PCR方法检测出外源青鱼GH基因在P0代转基因四倍体尾鳍基因组DNA中的整合率为90%;对20尾雄性P0代转基因四倍体精液样本的PCR检测发现,13个样本具有外源青鱼GH基因的整合.在一尾生长速度显著的P0代转基因四倍体鲫鲤的肌肉、肝脏、肾脏和卵巢组织中可检测到外源青鱼GH基因的转录.本研究成功获得了具有明显生长优势的P0代转青鱼GH基因异源四倍体鲫鲤,为建立转青鱼GH基因异源四倍体鲫鲤纯系和研制不育的转基因三倍体鱼奠定了基础.     

生长激素及其基因转移对鱼类生长和渗透压的调节作用

生长激素及其基因转移对鱼类生长和渗透压的调节起着重要作用。转生长激素基因鱼表现出明显的快速生长效应。生长激素促进了鲑科鱼类对海水的适应能力。本文对此进行了综述,并讨论了生长激素及其基因转移对鱼类生长和渗透压调节作用的生理机制、生长激素与胰岛素样生长因子以及甲状腺激素的关系。     

生长激素及其基因转移对鱼类生长和

生长激素及其基因转移对鱼类生长和渗透压的调节起着重要作用.转生长激素基因鱼表现出明显的快速生长效应.生长激素促进了鲑科鱼类对海水的适应能力.本文对此进行了综述,并讨论了生长激素及其基因转移对鱼类生长和渗透压调节作用的生理机制、生长激素与胰岛素样生长因子以及甲状腺激素的关系.     

转基因鱼

本文综述了转基因鱼研究的进展,并介绍了目的基因的选择及其导入的主要方法。此外,还对转基因鱼研究中尚存的一些问题作了说明。 自从八十年代中期开始鱼类基因转移研究以来,进展非常迅速。1985年Zhu等成功地将带有小鼠重金属结合蛋白(mMT)启动子的人生长激素(hGH)基因导入鲫鱼受精卵,并获得生长速度快的子代鱼。随后,世界一些国家开展了鱼类基因转移的研究,并取得很大成就。本文拟对转基因鱼研究现状及尚存的问题作一概述,供研究者参考。     

Gene transfer technology in aquaculture

The gene transfer technique, transgenesis, has permitted the transfer of genes from one organism to another to create new lineages of organisms with improvement in traits important to aquaculture. Genetically modified organisms (GMOs), therefore, hold promise for producing genetic improvements, such as enhanced growth rate, increased production and efficiency, disease resistance and expanded ecological ranges. The basic procedure to generate transgenic fish for aquaculture includes: (1) design and construction of transgenic DNA; (2) transfer of the gene construct into fish germ cells; (3) screening for transgenic fish; (4) determination of transgene expression and phenotype; (5) study of inheritance; and (6) selection of stable lines of transgenics.GMOs offer economic benefits, but also pose environmental threats. Optimising the mix of benefits and risks is of fundamental importance. The potential economic benefits of transgenic technology to aquaculture are obvious. Transgenic fish production has the goal of producing food for human consumption; thus the design of genetic constructs must take into consideration the potential risks to consumer health, as well as marketing strategies and product acceptance in the market.     

Expression of reporter genes introduced by microinjection and electroporation in fish embryos and fry.

The technique for foreign gene transfer in fish is becoming a novel method for genetic engineers to produce useful transgenic fish as well as for experimental purposes. Our studies of transgenic fish have been focused on methods of gene transfer and regulatory elements for transgene expression. I describe the characteristics of 3 gene transfer methods we have established (i.e., microinjection into fertilized eggs, microinjection into oocytes, and electroporation). Also described is a series of experiments to estimate activities of several promoters and enhancers in a fish cell line. Finally, experiments to examine activities of the elements shown to be active in the cell line in fish embryos and fry are described. SV2, miw, and metallothioneine promoters of trout and mouse were shown to be active in these experiments.     

Gene transfer in fish: Potential and practice

Abstract

During the past decade, developments in genetic engineering technology have led to the production of transgenic fish. In this paper, the impact of this novel strategy for genetic improvement of fish is examined in the context of possible benefits, probable limitations, and actual results. The role of gene transfer in improving fish growth rates, combatting disease, and altering other specific aspects of fish production is reviewed. Practical considerations, particularly in relation to the selection of a gene construct and its subsequent transfer, are outlined. A detailed discussion of the fate of transferred genes and the efficiency of transgenic fish production is also included. Finally, we review some of the practical precautions which must be observed if this novel technology is to safely complement conventional fish production strategies.     

Expression of a Novel Piscine Growth Hormone Gene Results in Growth Enhancement in Transgenic Tilapia (Oreochromis Niloticus)

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month old G2 transgenic fish is more than three fold that of their non transgenic siblings. Somewhat surprisingly G1 fish transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement, although salmon transgenic for this construct do show greatly enhanced growth. The growth enhanced transgenic lines were also strongly positive in a radio-immuno assay for the specific hormone in their serum, whereas the non growth enhanced lines were negative. Attempts to induce expression from the metallo thionein promoter by exposing fish to increased levels of zinc were also unsuccessful.Homozygous transgenic fish have been produced from the ocean pout antifreeze/chinook salmon GH construct and preliminary trials suggest that their growth performance is similar to that of the hemizygous transgenics. No abnormalities were apparent in the growth enhanced fish, although minor changes to skull shape and reduced fertility were noted in some fish. There is also preliminary evidence for improved food conversion ratios when growth enhanced transgenic tilapia are compared to their non-transgenic siblings.The long term objective of this study is to produce lines of tilapia which are both growth enhanced and sterile, so offering improved strains of this important food fish for aquaculture.     

Transgenic fish

Transgenic fish are produced by the artificial transfer of rearranged genes into newly fertilized eggs. Currently microinjection is the preferred method, although the integration rates of transgenes are generally low. A number of fusion genes, containing retrovirus sequences which direct integration, have been developed to enhance integration of transgenes. Mass gene transfer methods are also being developed. These include lipofection, particle bombardment, and electroporation of embryos and sperm cells. These methods are potentially useful for marine organisms such as crustaceans and molluscs as well as fish. In contrast to microinjection, which treats single cells individually, these methods can transfer genes into a large number of eggs at once. There is some evidence to indicate successful integration and expression of transgenes transferred by the electroporation of embryos and sperm cells. Germline transmission of transgenes has been observed through mating studies, and in some cases the progeny express the new phenotype consistently. However, germline transmission does not necessarily confirm stable integration of the transgene. There is evidence that transgenes may exist extrachromosomally. Transgenic fish are viewed as a useful model for the study of complex biological phenomena such as growth and differentiation, and as a fast track to the production of broodstock for the aquaculture industry. Current research focuses on the elucidation of the mechanisms controlling the regulation of gene expression. The use of transgenic fish for the isolation of developmental genes has just begun. Applications of transgenesis to broodstock development have been focused on the development of fish with accelerated growth, tolerance to low temperature, and disease resistance. However, before the release of transgenic fish into the environment, the possible impact on the environment must be assessed. There must be safeguards to protect the genetic diversities of the natural populations, and to conserve the natural habitats     

Integration mechanisms of transgenes and population fitness of GH transgenic fish

It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms of integration and gene targeting of the transgene, as well as the viability, reproduction and transgenic approaches for the reproductive containment of GH-transgenic fish. Further, we propose that it should be necessary to do the following studies, in particularly, of the breeding of transgeni...     

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.