Applications of avian transgenesis

The ability to introduce foreign DNA into the genome of an organism has proven to be one of the most powerful tools in modern biology. Methods for the manipulation of the animal genome have been developed at an impressive pace for 3 decades, but only in the past 5 years have useful tools for avian transgenesis emerged. The most efficient technique involves the use of replication-deficient lentiviral vectors to deliver foreign DNA into the avian germline. Although lentiviral-mediated transgenesis presents some constraints, progress in this area has garnered interest in both industry and academia for its potential applications in biological research, biotechnology, and agriculture. In this review we evaluate methods for the production of transgenic birds, focusing on the advantages and limitations of lentiviral-mediated transgenesis. We also provide an overview of future applications of this technology. The most exciting of these include disease-resistant transgenic poultry, genetically modified hens that produce therapeutic proteins in their eggs, and transgenic songbirds that serve as a model to study communication disorders. Finally, we discuss technological advances that will be necessary to make avian transgenesis a more versatile tool.     

Avian embryos and related cell lines: A convenient platform for recombinant proteins and vaccine production

Chick embryos are a significant historical research model in basic and applied sciences. The embryonated eggs have been used for virus inoculation in order to vaccine production for nearly a century. Recently, avian eggs and cell lines derived from embryonated eggs have found wide application in biotechnology. This review will discuss about the unique characteristics of avian eggs in terms of safety, large scale and economical production of recombinant proteins. This system also provides the human‐like glycosylation on target proteins and therefore can be considered as a suitable host for biomanufacturing of humanized monoclonal antibodies and therapeutic proteins. Avian derived cell lines are an alternative for rapid vaccine manufacturing during a pandemic. Based on the latest knowledge in cell and animal transgenesis, the currently available germ cell‐mediated gene transfer system provides a more efficient strategy in gene targeting and creation of transgenic birds that lead to advancements in industrial, biotechnology, and biological research applications. This review covers the recent development of avian fertilized eggs and related cell lines in a variety of human biopharmaceuticals and viral vaccine manufacturing.     

植物遗传转化表达载体研究进展

植物遗传转化表达载体是植物转基因研究中非常重要的一个环节,外源基因在转基因植物中的高效表达是转基因研究成功的关键。综述了植物遗传转化表达载体近年来的研究进展情况,着重介绍了在转基因植物中实现外源基因高效表达的多种途径和策略,旨在提高转基因植物中外源基因的表达水平和生物安全性,并展望了今后植物转基因研究及商业化发展方向。     

TECHNICAL ADVANCES: A microarray for large-scale genomic and transcriptional analyses of the zebra finch (Taeniopygia guttata) and other passerines

The microarray technology has revolutionized biological research in the last decade. By monitoring the expression of many genes simultaneously, microarrays can elucidate gene function, as well as scan entire genomes for candidate genes encoding complex traits. However, because of high costs of sequencing and design, microarrays have largely been restricted to a few model species. Cross-species microarray (CSM) analyses, where microarrays are used for other species than the one they were designed for, have had varied success. We have conducted a CSM analysis by hybridizing genomic DNA from the common whitethroat (Sylvia communis) on a newly developed Affymetrix array designed for the zebra finch (Taeniopygia guttata), the Lund-zf array. The results indicate a very high potential for the zebra finch array to act as a CSM utility in other passerine birds. When hybridizing zebra finch genomic DNA, 98% of the gene representatives had higher signal intensities than the background cut-off, and for the common whitethroat, we found the equivalent proportion to be as high as 96%. This was surprising given the fact that finches and warblers diverged 25-50 million years ago, but may be explained by a relatively low sequence divergence between passerines (89-93%). Passerine birds are widely used in studies of ecology and evolution, and a zebra finch array that can be used for many species may have a large impact on future research directions.     

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.     

Efficient production of germline transgenic chickens using lentiviral vectors

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.     

Identification of transgene integration site and anatomical properties of fluorescence intensity in a EGFP transgenic chicken line

Transgenic birds are commonly used for time-lapse imaging and fate mapping studies in developmental biology. When researchers use transgenic birds expressing fluorescent protein, they need to understand the integration site of the transgene in the genome and the intensity of fluorescence in the tissues of interest. In this study, we determined the integration site of the transgene and fluorescence property of developing organs in our transgenic chicken line generated by lentivirus infection. The transgene was localized between exons 3 and 4 of MED27. Some homozygotes and heterozygotes appeared to be lethal at early embryonic stages. We performed histological analysis of EGFP expression in transgenic embryos at St. 14, 17, and 24 by immunohistochemistry with anti-GFP antibody on paraffin sections. Next, we cut cryosections and quantified direct EGFP intensity from the transgene in each tissue without performing immunohistochemistry. These results revealed that EGFP intensity in each tissue was unique in developing embryos and changed according to developmental stages. Finally, we demonstrated that EGFP-expressing cells in a micromass culture with co-culturing wild-type cells were clearly distinguishable via live cell imaging. These results provide essential information on the potential of our transgenic line and indicate that these transgenic chicken lines are useful for research associated with developmental biology.     

精子介导法建立sFat-1转基因小鼠模型

利用精子介导的基因转移(Sperm-mediated gene transfer,SMGT)方法,将sFat-1 DNA片段直接和小鼠精子孵育,再通过体外受精、胚胎移植等技术建立sFat-1转基因小鼠模型。经PCR检测,16只后代中发现2只阳性个体,Southern blot进一步鉴定的结果显示有2只后代整合了sFat-1基因,成功建立sFat-1转基因小鼠模型,转基因率为12.5%。本研究运用精子介导的转基因方法将sFat-1基因整合到小鼠基因组中,为进一步研究sFat-1的生物学功能提供了实验动物模型。     

紫花苜蓿转基因技术及其应用的研究进展

对农杆菌介导法、电击法、显微注射法及基因枪法等几种常用的紫花苜蓿转基因技术研究进展进行概述。综述利用转基因技术对苜蓿进行品质改良,提高抗逆性、抗病虫害、抗除草剂,以及利用转基因苜蓿作为生物反应器生产植物疫苗、酶制剂和抗体等方面的研究进展及最新研究成果。对目前制约紫花苜蓿转基因技术发展的瓶颈及其生物安全性问题进行分析和讨论,并对其应用前景和商业价值进行展望。     

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology¹. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.     

“What’s wrong with my monkey?” Ethical perspectives on germline transgenesis in marmosets

The birth of the first transgenic primate to have inherited a transgene from its parents opens the possibility to set up transgenic marmoset colonies, as these monkeys are small and relatively easy to keep and breed in research facilities. The prospect of transgenic marmoset models of human disease, readily available in the way that transgenic laboratory mice are currently, prompts excitement in the scientific community; but the idea of monkeys being bred to carry diseases is also contentious. We structure an ethical analysis of the transgenic marmoset case around three questions: whether it is acceptable to use animals as models of human disease; whether it is acceptable to genetically modify animals; and whether these animals’ being monkeys makes a difference. The analysis considers the prospect of transgenic marmoset studies coming to replace transgenic mouse studies and lesion studies in marmosets in some areas of research. The mainstream, broadly utilitarian view of animal research suggests that such a transition will not give rise to greater ethical problems than those presently faced. It can be argued that using marmosets rather than mice will not result in more animal suffering, and that the benefits of research will improve with a move to a species more similar in phylogenetic terms to humans. The biological and social proximity of monkeys and humans may also benefit the animals by making it easier for scientists and caretakers to recognize signs of suffering and increasing the human motivation to limit it. The animal welfare and research impacts of the transition to marmoset use will depend very much on the extent to which researchers take these issues seriously and seek to minimize animal harm and optimize human benefit.     

Making better transgenic models

Over the last decade transgenic mouse models have become a common experimental tool for unraveling gene function. During this time there has been a growing expectation that transgenes resemble the in vivo state as much as possible. To this end, a preference away from heterologous promoters has emerged, and transgene constructs often utilize the endogenous promoter and gene sequences in BAC, PAC and YAC form without the addition of selectable markers, or at least their subsequent removal. There has been a trend toward controlled integration by homologous recombination, either at a characterized chromosomal localization or in some cases within the allele of interest. Markers such as green fluorescent protein (GFP), beta-galactosidase (LacZ), and alkaline phosphatase (AP) continue to be useful to trace transgenic cells, or transgene expression. The development of technologies such as RNA interference (RNAi), are introducing new ways of using transgenic models. Future developments in RNAi technology may revolutionize tissue specific inactivation of gene function, without the requirement of generating conditionally targeted mice and tissue specific recombinase mice. Transgenic models are biological tools that aid discovery. Overall, the main consideration in the generation of transgenic models is that they are bona fide biological models that best impart the disease model or biological function of the gene that they represent. The main consideration is to make the best model for the biological question at heart and this review aims to simplify that task somewhat. Here we take a historical perspective on the development of transgenic models, with many of the important considerations to be made in design and development along the way.     

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology.     

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。     

A habitat island approach to conserving birds in urban landscapes: case studies from southern and northern Europe

Wildlife conservation in urban habitats is increasingly important due to current urbanization trends. We review the different approaches to studying birds in urban landscapes, and point out the importance of the habitat island ecological theory as a research framework for the management and conservation of urban birds. Based on two comprehensive research projects conducted at urban parks in Spain (Madrid) and Finland (Oulu and Rovaniemi), several different issues related to bird conservation in cities are discussed, main findings of these projects are presented, and future research needs are suggested. Urban parks are important biodiversity hotspots in cities. Fragmentation conditions have the same deleterious effects to urban birds as in other fragmented landscapes. Park size accounts for species accumulation in urban parks; this pattern being highly nested. Urban parks of 10–35 ha would contain most of the species recorded in cities, but other indicators related to the probabilities of persistence of the target species should be obtained. Wooded streets can increase urban landscape connectivity by providing alternative habitat for feeding and nesting during the breeding season. Because increasing the size of parks is difficult in cities, enhancement of habitat diversity and resource availability for birds within parks (e.g. nest boxes, winter feeding tables, etc.) appears to be a straightforward way of increasing urban bird diversity. However, human disturbance (pedestrians) should be controlled since it can negatively influence many urban birds. We present a conceptual model for urban bird conservation, which includes three aspects (management, environmental education and research) and new alternatives to promote the involvement of different sectors of the society.     

Der Zebrafisch als vielseitiges Modellsystem. Vom Zierfisch zum Forschungsobjekt

Over the past years, the zebrafish rose from an aquarium fish to a widespread model of biomedical research. It is easily maintained, produces relatively large offspring, shows rapid extra maternal development, and is transparent during early development. Hence, it was initially primarily utilized for direct microscopic observation of developmental processes. However, as it is easily genetically manipulated, it was soon used to produce transgenic lines expressing DNA derived from other organisms. These lines could be used to study the importance of specific genes for biological processes. In addition, zebrafish is used for random mutagenesis screens, allowing the unbiased detection of genes relevant for diseases, for small molecule screens applied for the discovery of biologically active substances, and increasingly as a model for cancer research.     

The zebrafish as a model system in developmental, toxicological and transgenic research

The zebrafish has long been used as a model system in fisheries biology and toxicology. More recently, it has also become the focus of a major research effort into understanding the molecular and cellular events which dictate the development of vertebrate embryos. As well, the zebrafish has proven attractive in studies examining the factors which affect the creation of transgenic fish and the expression of transgenes. The advances which have been made in these areas have firmly established this small aquarium fish as a major model system in biological and biotechnological research.     

The natural link between Europe and Africa – 2.1 billion birds on migration

The Palaearctic–African migration system comprises enormous numbers of birds travelling between Europe and Africa twice each year. Migratory birds may form strong links between the two continents given they can act as both transport vehicles for parasites and diseases as well as temporary consumers with increased food demand to fuel their flight. Knowing the number of migrating birds is crucial if such links are to be quantified. We estimate that today approximately 2.1 billion songbirds and near-passerine birds migrate from Europe to Africa in autumn, 73% of which are accounted for by just 16 species. This number is only half the estimate from the 1950s in the only other assessment to date. The discrepancy is mainly caused by the limited information on population sizes in the past. Our estimated number of migrants is highly dependent on the accuracy of the underlying estimates of breeding population sizes, as well as breeding parameters in species with relatively high reproductive output. The updated figures quantify and emphasize the strong natural connection between Africa and Europe, which has important implications for manifold research topics including those related to climate change, human health and biological conservation.     

Medaka as a model of transgenic fish.

The medaka (Oryzias latipes) is an egg-laying fresh-water fish. We describe the medaka as a model system of transgenic fish in germs of biological characteristics, manipulation of embryos, gene expression in development, and basic research in aquaculture. The fish are small (approximately 3 cm in length) and have a short generation time (approximately 3 months). The eggs are easy to manipulate. A foreign gene (e.g., the chicken delta crystallin gene) is transferred and expressed stage-dependently in development of medaka embryos. Growth hormone genes of vertebrates are transferred and expressed and, in some cases, accelerate growth of the fish. Thus, the medaka is one of the most promising models of transgenic fish for basic research of gene expression and aquaculture.