转基因技术在水产动物营养中的应用

近年来,随着分子生物学研究的进展,转基因技术得到了迅猛的发展,在水产动物营养中也有广泛的应用.动物转基因技术是指将外源基因或体外重组的基因结构导入动物的基因组内,使其在动物体内整合和表达,产生具有新的遗传性状,并能将这种性状稳定地传递给后代的一种技术.对转基因技术的一般方法,如显微注射、精子载体、电穿孔,逆转录病毒载体感染和基因枪等方法,以及转基因技术在水产动物营养中的应用进行了综述.     

外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

动物转基因新技术研究进展

动物转基因技术是21世纪发展最为迅速的生物高新技术之一, 它是指通过基因工程技术将外源基因整合到受体动物基因组中, 从而使其得以表达和遗传的生物技术。动物转基因的关键限制因素是转基因效率和基因表达的精确调控。目前有多种转基因技术, 每一种技术各有其优缺点, 仍然需要进一步研究。随着研究的深入, 转基因技术必将在探讨基因功能、动物遗传改良、生物反应器、动物疾病模型、器官移植等领域有广阔的应用前景。文章综述了近年发展的提高转基因效率的生殖干细胞法、提高转基因精确性的基因打靶法、RNA干扰(RNAi)介导的基因沉默技术和诱导多能干细胞(iPS)转基因技术。新的转基因技术为转基因动物的研究提供了更好的平台, 可以加快促进人类医药卫生、畜牧生产等领域的发展。     

体细胞基因打靶制备动物乳腺生物反应器的策略与应用

在转基因动物研究中,由于基因表达调控元件的人工拼接和外源基因在动物基因组中随机整合所带来的“位置效应”,致使转基因动物外源基因的表达水平不高并且差异较大。为此,利用定位整合优势,对以基因同源重组为基础的基因打靶技术进行了大量研究。介绍了就利用体细胞基因打靶和核移植技术制备动物乳腺生物反应器的策略和应用情况做一综述,并对提高基因打靶效率的各种策略,打靶细胞的选择,转基因细胞核移植的低融合事件以及基因打靶制备乳腺生物反应器的优越性进行分析。     

促绒毛生长基因及其在转基因动物中的应用

通过转基因克隆技术使得外源基因在皮肤细胞内超表达,进而促进毛囊发育和绒毛生长是培育高产量绒毛动物品系的有效途径.在转基因动物中构建真核表达栽体常用的皮肤特异性启动子主要是皮肤角蛋白基因启动子,具有促绒毛生长功能的基因则包括编码生长因子、转录因子及小分子蛋白的基因.目前已获得利用这些特异性启动子调控外源基因在皮肤细胞内超表达的转基因动物.     

转基因植物外源基因的整合分析

外源基因表达的不稳定性和多样性是制约转基因作物育种研发进度的关键因素,外源基因的整合情况与外源基因的表达直接相关,充分了解外源基因的整合情况可为构建高效表达载体、获得外源基因稳定一致表达转基因材料提供参考,同时为转基因作物的安全利用提供保障.就外源基因整合情况的分析方法、不同转化方法外源基因的整合特点及利用定点整合技术提高外源基因表达稳定一致性的研究进展作一概述.     

转基因动物及其在生物反应器中的应用与市场现状

转基因动物是指用DNA重组技术将人们所需要的目的基因导入动物的受精卵或早期胚胎内,使外源目的基因随细胞的分裂而增殖并在体内表达,且能稳定地遗传给后代的动物。1982年美国华盛顿大学Palmiter等将大鼠生长激素基因导入小白鼠的受精卵里得到比正常体格大一倍的“超级小鼠”,此成果带动了转基因动物的一系列研究。     

采用高分子介导精子作载体制备转基因泥鳅

为探讨树形高分子介导精子载体技术产生转基因动物,将泥鳅精子与具有标记基因LacZ的pCH110重组质粒和树形高分子在保存液内孵育,经DNA原位杂交检测发现树形高分子介导下精子携事外源DNA的效率得到较大的提高,将捕获了外源DNA的精子,再与泥鳅卵进行体外人工受精。由此发育的鱼苗经PCR和LacZ组织化学检测,获得了高比例的转基因泥鳅,外源基因LacZ在泥鳅幼苗头部得到了明显表达。     

转基因动物与基因表达调控的研究

十余年来,随着转基因技术的快速发展,基因表达调控的转基因研究已逐渐开展。本文在下列几方面介绍了转基因动物技术在基因表达调控中的应用。转基因动物可作为在体内研究外源基因表达调控的“反应器”,例如研究酶对底物作用的种属特异性。可以精确研究ＤＮＡ顺式作用序列,探讨包括改变基因表达在内的组织和阶段特异性表达,增强子、内含子和核基质附着区的调节作用以及各基因间相互作用。转基因动物可研究发育中的时、空调节（如     

SND1转基因小鼠的构建

目的:构建 SND1 过表达的转基因小鼠模型。方法:利用对小鼠 SND1 基因转录本构建 SND1 过表达载体pInsulator-CAG-3×FLAG-SND1,利用受精卵原核注射技术,将外源线性pInsulator-CAG-3×FLAG-SND1转基因载体注射到受精卵细胞核内,将存活受精卵进行胚胎移植制备 SND1 转基因小鼠,用PCR、RT-PCR技术鉴定转基因小鼠是否构建成功。结果:成功构建过表达 SND1 基因的转基因小鼠模型,为进一步研究 SND1 基因在动物体内的生物学功能奠定基础。     

Artificial chromosome transgenesis in pigmentary research

Pigmentary genes were among the first mammalian genes to be studied, mostly because of the obvious phenotypes associated with their mutations. In 1990, tyrosinase, encoding the limiting enzyme in the melanin synthesis pathway, was eventually assigned to the c (albino) locus by classical rescue experiments driven by functional constructs in transgenic mice. These pioneer reports triggered the study of the regulation of endogenous tyrosinase gene expression by combining different amounts of upstream regulatory and promoter regions and testing their function in vivo in transgenic animals. However, faithful and reproducible transgenic expression was not achieved until the entire tyrosinase expression domain was transferred to the germ-line of mice using artificial-chromosome-type transgenes. The use of these large tyrosinase transgenic constructs and the ease with which they could be manipulated in vitro enabled the discovery of previously unknown but fundamental regulatory regions, such as the tyrosinase locus control region (LCR), whose presence was required in order to guarantee position-independent and copy-number-dependent expression of tyrosinase transgenes, with an expression level, per copy, comparable to that of an endogenous wild-type allele. Subsequently, functional dissection of elements present within this LCR through the generation of new artificial-chromosome type tyrosinase transgenes has revealed the existence of different regulatory activities. The existence of some of these units had been suggested previously by standard-type transgenic analyses. In this review, we will discuss both independent approaches and conclude that optimal tyrosinase transgene expression requires the use of its complete expression domain.     

Size Matters: Use of YACs,BACs and PACs in Transgenic Animals

In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.     

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation.

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.     

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.     

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization. Received: 20 December 2001 / Revision accepted: 6 June 2001