Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection

The low efficiency of current microinjection-based animal transgenesis techniques is largely the result of poor embryo survival. We have developed a new, bacterial recombinase-based transgenesis method. Intracytoplasmic sperm injection (ICSI) of single stranded DNA (ssDNA) complexed with E. coli recombinase RecA into mouse metaphaseII (MII) arrested oocytes resulted in RecA-dependent transgenesis. This approach offers significant advantages over pronuclear microinjection and previous ICSI-based transgenesis approaches in terms of improved embryo survival, which translates into greater transgenesis efficiency. It also opens the possibility to attempt experiments, which may affect gene targeting by homologous recombination into DNA of mammalian single celled pre-implantation embryos.     

Lentiviral transgenesis

Transgenic animals are relevant for many fields of modern biomedicine and agriculture. However, the inefficiencies of the presently available techniques – DNA microinjection and retroviral gene transfer – have led to an explosion of costs for transgenics especially in farm animals. The recent success in transferring genes to early embryos of different species (mouse, rat, pig, cattle) by viral vectors derived from lentiviruses, has established lentiviral transgenesis as an exciting alternative to the classical method of DNA microinjection. In addition, lentiviral vectors can be used to transfer genes into embryonic stem cells. Due to its high efficacy and versatility, lentiviral transgenesis should have a big impact on transgenic research.     

Transplantation of a polyembryonic wasp embryo: a technique for transferring endoparasitic embryo into the host egg

Concealed development of many animal embryos prevents examination of development and limits the application of embryo manipulation techniques aimed at understanding developmental processes. In embryos developing in utero, such as in mammals, it is necessary to dissect embryos from the mother and, upon manipulative intervention, to implant them back into the recipient. Parasitic wasps present a promising system for understanding the evolution of early developmental processes. In basal ectoparasitic species that lay eggs on the surface of the host, it is possible to adapt embryo manipulation techniques developed in Drosophila. However, their derived endoparasitic relatives, which exhibit various modifications of developmental programs, undergo concealed development within the host body. For example, the parasitic polyembryonic wasp Copidosoma floridanum oviposits an egg into the egg of the host moth Trichoplusia ni. The host larva emerges and the parasite undergoes development within the host body, preventing embryo manipulation as a means of examining developmental regulation. Here we present a protocol for embryo transfer that allows the transplantation of C. floridanum egg into the host egg. This approach opens a new avenue in the application of various embryo manipulation techniques aimed at understanding the evolution of embryogenesis in endoparasitic Hymenoptera. In addition, this approach has potential for the development of other tools in C. floridanum, such as transgenesis and reverse genetics, which can also be extended to other endoparasitic species.     

Relations between animal transgenesis and reproduction

Transgenesis has become an essential tool for the study of gene expression mechanisms and functions. Transgenesis is also more and more used for biotechnological applications such as the study of human diseases, the adaptation of pig organs to humans, the production of pharmaceutical proteins in milk and likely in the future for the improvement of animal production. The use of transgenesis relies on the efficiency of gene transfer. New tools have been recently designed to improve gene transfer. The methods of gene transfer are highly dependent on the techniques of animal reproduction. Conversely, the need to improve transgenesis urges researchers to study some of the key steps in reproduction and to find new techniques for gene transfer. This paper summarises the recent data and the perspectives offered by animal transgenesis.     

Active integration: new strategies for transgenesis

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed “Active Transgenesis” to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac’s specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.     

Minos transposon causes germline transgenesis of the ascidian Ciona savignyi

An ascidian, Ciona savignyi, is regarded as a good experimental animal for genetics because of its small and compact genome for which a draft sequence is available, its short generation time and its interesting phylogenic position. ENU-based mutagenesis has been carried out using this animal. However, insertional mutagenesis using transposable elements (transposons) has not yet been introduced. Recently, one of the Tc1/mariner superfamily transposons, Minos, was demonstrated to cause germline transgenesis in the related species Ciona intestinalis. In this report, we show that Minos has the ability to transpose from DNA to DNA in Ciona savignyi in transposition assays. Although the activity was slightly weaker than in Ciona intestinalis, Minos still caused germline transgenesis in Ciona savignyi. In addition, one insertion seemed to have caused an enhancer trapping. These results indicate that Minos provides a potential tool for transgenic techniques such as insertional mutagenesis in Ciona savignyi.     

Animal transgenesis: state of the art and applications

There is a constant expectation for fast improvement of livestock production and human health care products. The advent of DNA recombinant technology and the possibility of gene transfer between organisms of distinct species, or even distinct phylogenic kingdoms, has opened a wide range of possibilities. Nowadays we can produce human insulin in bacteria or human coagulation factors in cattle milk. The recent advances in gene transfer, animal cloning, and assisted reproductive techniques have partly fulfilled the expectation in the field of livestock transgenesis. This paper reviews the recent advances and applications of transgenesis in livestock and their derivative products. At first, the state of art and the techniques that enhance the efficiency of livestock transgenesis are presented. The consequent reduction in the cost and time necessary to reach a final product has enabled the multiplication of transgenic prototypes around the world. We also analyze here some emerging applications of livestock transgenesis in the field of pharmacology, meat and dairy industry, xenotransplantation, and human disease modeling. Finally, some bioethical and commercial concerns raised by the transgenesis applications are discussed.     

蜜蜂转基因研究进展及研究途径探讨

转基因动物的科研价值和商业价值促进了转基因技术的不断发展和在各个领域的深入应用。蜜蜂是有着悠久饲养历史的经济昆虫和在基础理论研究领域有重大应用价值的模式动物,但其转基因研究却相对落后。雌性蜂的级型分化和工蜂清洁巢房行为增加了蜜蜂转基因的难度,精子介导转基因配套以人工授精技术及蜜蜂卵或幼虫的转基因操作与蜜蜂人工孵育技术结合是目前蜜蜂转基因的较好途径。文章综述蜜蜂转基因的研究进展,并讨论蜜蜂转基因所面临的特殊性及其研究途径。     

Gene transfer into chicken embryos as an effective system of analysis in developmental biology

Chicken embryos have been used as a model animal in developmental biology since the time of comparative and experimental embryology. Recent application of gene transfer techniques to the chicken embryo increases their value as an experimental animal. Today, gene transfer into chicken cells is performed by three major systems, lipofection, electroporation and the virus-mediated method. Each system has its own features and applicability. In this overview and the associated four minireviews, the methods and application of each system will be presented.     

Generation and characterization of a GFP transgenic rat line for embryological research

Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103 surviving embryos resulted in the production of 35 offspring (33.9%) and two of them were transgenic (5.7%). Two transgenic rat lines that ubiquitously express GFP under the control of the cytomegalovirus-enhancer/beta-actin (CAGGS) promoter were generated by breeding. We studied the main embryological parameters of one these GFP transgenic lines. Homozygous GFP-transgenic females have the same ovulation and superovulation rates as wild type (WT) females. Transgenic embryos reached blastocyst stage in vitro and developed in vivo after embryo transfer without decrease in their developmental ability compared to the control group. The genotype of the parents determined the onset of GFP expression in preimplantation embryos. When the GFP gene is derived from the transgenic female parent, fluorescence was detected in oocytes and in embryos of all further stages of development. When the GFP gene is inherited by the transgenic male parent, GFP was only expressed from the blastocyst stage on. GFP-transgenic rats represent a valuable tool to mark embryos for many embryological studies such as transgenesis, gene expression patterns during early development, embryo aggregation for analysis of the distribution of cells in chimeric embryos and nuclear transfer to confirm the origin of the cloned offspring.     

The Meganuclease I-SceI Containing Nuclear Localization Signal (NLS-I-SceI) Efficiently Mediated Mammalian Germline Transgenesis via Embryo Cytoplasmic Microinjection

The meganuclease I-SceI has been effectively used to facilitate transgenesis in fish eggs for nearly a decade. I-SceI-mediated transgenesis is simply via embryo cytoplasmic microinjection and only involves plasmid vectors containing I-SceI recognition sequences, therefore regarding the transgenesis process and application of resulted transgenic organisms, I-SceI-mediated transgenesis is of minimal bio-safety concerns. However, currently no transgenic mammals derived from I-SceI-mediated transgenesis have been reported. In this work, we found that the native I-SceI molecule was not capable of facilitating transgenesis in mammalian embryos via cytoplasmic microinjection as it did in fish eggs. In contrast, the I-SceI molecule containing mammalian nuclear localization signal (NLS-I-SceI) was shown to be capable of transferring DNA fragments from cytoplasm into nuclear in porcine embryos, and cytoplasmic microinjection with NLS-I-SceI mRNA and circular I-SceI recognition sequence-containing transgene plasmids resulted in transgene expression in both mouse and porcine embryos. Besides, transfer of the cytoplasmically microinjected mouse and porcine embryos into synchronized recipient females both efficiently resulted in transgenic founders with germline transmission competence. These results provided a novel method to facilitate mammalian transgenesis using I-SceI, and using the NLS-I-SceI molecule, a simple, efficient and species-neutral transgenesis technology based on embryo cytoplasmic microinjection with minimal bio-safety concerns can be established for mammalian species. As far as we know, this is the first report for transgenic mammals derived from I-SceI-mediated transgenesis via embryo cytoplasmic microinjection.     

HUMAN DIGNITY IN INTERNATIONAL POLICY DOCUMENTS: A USEFUL CRITERION FOR PUBLIC POLICY?

Current developments in biomedicine are presenting us with difficult ethical decisions and raising complex policy questions about how to regulate these new developments. Particularly vexing for governments have been issues related to human embryo experimentation. Because some of the most promising biomedical developments, such as stem cell research and nuclear somatic transfer, involve such experimentation, several international bodies have drafted documents aimed to provide guidance to governments when developing biomedical science policy. Here I focus on two such documents: the Council of Europe's Convention for the Protection of Human Rights and Dignity of the Human Being and the Additional Protocol to the Convention for the Protection of Human Rights and Dignity of the Human Being. I argue that by using human dignity as a criterion to determine the permissibility of particular human embryo research practices, these documents cannot aid in identifying research that would be contrary to human dignity. Thus, they fail to guide public policy on embryo experimentation. Their use of human dignity as a criterion makes their task of offering guidance unfeasible because the concept as used in these documents is too vague and is applied in contradictory ways. I discuss the main goals of these documents and their claims in relation to human embryo research. I then discuss how they have influenced public policy in several countries. Finally, I show that although these Council of Europe treaties attempt to serve as public policy guides in the area of embryo research, they fail to do so.     

Progress and biotechnological prospects in fish transgenesis

The history of transgenesis is marked by milestones such as the development of cellular transdifferentiation, recombinant DNA, genetic modification of target cells, and finally, the generation of simpler genetically modified organisms (e.g. bacteria and mice). The first transgenic fish was developed in 1984, and since then, continuing technological advancements to improve gene transfer have led to more rapid, accurate, and efficient generation of transgenic animals. Among the established methods are microinjection, electroporation, lipofection, viral vectors, and gene targeting. Here, we review the history of animal transgenesis, with an emphasis on fish, in conjunction with major developments in genetic engineering over the past few decades. Importantly, spermatogonial stem cell modification and transplantation are two common techniques capable of revolutionizing the generation of transgenic fish. Furthermore, we discuss recent progress and future biotechnological prospects of fish transgenesis, which has strong applications for the aquaculture industry. Indeed, some transgenic fish are already available in the current market, validating continued efforts to improve economically important species with biotechnological advancements.     

Embryo manipulation in research and animal production

Research in developmental biology has resulted in techniques to accelerate changes in gene frequency and to interfere directly in the genome. Procedures already in use or being adapted to livestock include embryo transfer, chimera production, embryo splitting, gene transfer and nuclear transplantation. Experiments with mouse embryos are revealing the principles governing embryonic development and differentiation and illustrate the need for these investigations to be extended to embryos of livestock. The optimal combination of these technologies in animal production strategies will depend upon further research and the role of animal products in society.     

The chick; a great model system becomes even greater

The chick embryo has a long and distinguished history as a major model system in developmental biology and has also contributed major concepts to immunology, genetics, virology, cancer, and cell biology. Now, it has become even more powerful thanks to several new technologies: in vivo electroporation (allowing gain- and loss-of-function in vivo in a time- and space-controlled way), embryonic stem (ES) cells, novel methods for transgenesis, and the completion of the first draft of the sequence of its genome along with many new resources to access this information. In combination with classical techniques such as grafting and lineage tracing, the chicken is now one of the most versatile experimental systems available.     

Reproductive biotechnology and "big" biological questions

In recent decades, scientists have learned to manipulate that cardinal characteristic of life, reproduction, with powerful techniques like artificial insemination, contraception, embryo transfer, cryopreservation, and cloning by nuclear transfer. While these technologies often are used for practical applications and basic research, they have another profound intrinsic quality, which is to engender deep-seated thinking about important biological questions. Examples that stimulate such thinking include a goat's giving birth to her identical twin sister via splitting embryos, cryopreservation, and embryo transfer; that a parthenogenetic embryo can never become an animal but can become a genetic mother via an aggregation chimera; or that a somatic cell can become the sole genetic parent of a calf via cloning. In this paper, I illustrate this thought-stimulating quality by considering contributions of reproductive technologies to understanding, if not completely answering, several important biological questions.     

Review of scientific contributions by the Belgian medical centers concerned with human in vitro fertilization and embryo transfer (IVF).

In vitro fertilization and embryo transfer (IVF) may be considered as a particular application of modern medical therapeutics linked to human reproduction. The treatment of human sterility therefore involves some fundamental human values such as life, love and death. The quality of this highly technological treatment with fast knowledge of outcome at the end of the patient's menstrual cycle has been evaluated since the early 80s. It is a typically multidisciplinary team effort involving medical doctors, biologists, laboratory technicians, nurses and clerks that is representative of modern medical practice. IVF covers much more than just embryology, as this review will explain. IVF developed in close relation with clinical and experimental research protocols, which are the major topics of this paper. The newness of the techniques used led to the necessary interactions between clinicians and biologists working on animal experimental embryology.     

Efficient production of transgenic mice by intracytoplasmic injection of streptolysin‐O‐treated spermatozoa

Many methods for efficient production of transgenic animals for biomedical research have been developed. Despite great improvements in transgenesis rates resulting from the use of intracytoplasmic sperm injection (ICSI), the ICSI‐based sperm‐mediated gene‐transfer (iSMGT) technique is still not optimal in terms of sperm permeabilization efficiency and subsequent development. Here, we demonstrate that streptolysin‐O (SLO) can efficiently permeabilize mouse spermatozoa, leading to improved developmental competence and high transgenesis rates in iSMGT embryos and pups. In particular, the most efficient production of iSMGT‐transgenic embryos resulted from pretreatment with 5 U/ml SLO for 30 min and co‐incubation with 1.0 ng/µl of an EGFP expression vector. By incubating spermatozoa with Cy‐3‐labelled DNA, we found that fluorescence intensity was prominently detected in the head region of SLO‐treated spermatozoa. In addition, blastocyst development rate and blastomere survival were greatly improved by iSMGT using SLO‐treated spermatozoa (iSMGT‐SLO) as compared to freeze‐thawed spermatozoa. Consistent with this, a high proportion of transgenic offspring was obtained by iSMGT‐SLO after transfer into foster mothers, reaching 10.6% of the number of oocytes used (42.3% among pups). Together with successful germline transmission of transgenes in all founders analyzed, our data strongly suggest that SLO makes spermatozoa amenable to exogenous DNA uptake, and that the iSMGT‐SLO technique is an efficient method for production of transgenic animals for biomedical research. Mol. Reprod. Dev. 80: 233–241, 2013. © 2013 Wiley Periodicals, Inc.     

Optimized transgenesis in Xenopus laevis/gilli isogenetic clones for immunological studies

Xenopus laevis provides a unique animal model, alternative to mouse, to study immunology. Even though, several methodologies have been developed for the generation of transgenic Xenopus, to date none have been adapted for the X. laevis/gilli (LG) isogenetic clones that are essential for immunological studies. Since LG clones are generated via gynogenesis, transgenic methods using transgene integration into the sperm nuclei are not suited. Therefore, we have tested three alternative methods for LG transgenesis: the phiC31 integrase, the Sleeping Beauty transposase, and the I-SceI meganuclease. All three techniques produced transgenic LG clones; however, the I-SceI meganuclease was most effective. It resulted in high transgenesis efficiency (35-50%), bright nonmosaic GFP expression as well as stable germline transmission with 100% of the progeny carrying the transgene. Production of transgenic LG clones will allow us to modulate immune gene expression and further strengthen X. laevis as a biomedical model.