Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT)or female germ cell mediated gene transfer(FGCMGT)technique.Sperm-mediated gene transfer (SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been extensively used for producing transgenic animals.The newly developed approach of FGCMGT,ovary-mediated gene transfer(OMGT) is also a novel and useful tool for efficient transgenesis.This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques,methods developed and mechanisms of nucleic acid uptake by germ cells.     

YAC transgenesis in farm animals: Rescue of albinism in rabbits

The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis—such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene—have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation. © 1996 Wiley-Liss, Inc.     

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.     

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.     

Polyplex‐mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age‐related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome‐mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex‐mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20\% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex‐mediated gene transfer, but stable integration does occur at low frequencies with and without selection. J. Cell. Biochem. 76:153–160, 1999. © 1999 Wiley‐Liss, Inc.     

Transgenesis and nuclear transfer using porcine embryonic germ cells

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.     

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.     

精子介导转基因动物的制备

近年来 ,通过显微注射DNA至孵育的卵母细胞原核或外源基因转染后的胚胎干细胞进行转基因动物的生产已取得了令人瞩目的成就。在过去的 1 0年中 ,以精子作载体制备转基因哺乳动物或脊椎动物也取得了一些不同程度的进展。这些技术主要包括 :直接将外源DNA与精子共孵育至成熟 ;提取分离的精子DNA或进行预处理至精子发育成熟 ;以及在辅助受精前分离精子细胞等。此外 ,一些显微注射技术 ,如在输精管内进行体内直接转染雄性生殖细胞 ;将体内转染的雄性生殖细胞植入已分离的雄性生殖细胞 ,再显微注射至受体的睾丸 ,这些技术也逐渐成熟起来。研究表明 ,通过体内、体外转染外源DNA的显微操作技术只需将雄性受体与野生型雌性交配就可产生出转基因的后代个体 ,同时也避免了辅助受精和胚胎操作带来的机械损伤 ,因此具有一定的优势。本文综述了精子介导转基因 (SMGT)技术的发展历程、研究现状及前沿进展。     

Enhanced transgenesis by intracytoplasmic injection of envelope-free lentivirus

We demonstrate enhanced transgenesis in mice by intracytoplasmic injection of envelope-free lentivirus. Envelope-free lentivirus carrying the green fluorescent protein (GFP) gene under the control of the ubiquitin promoter (LVU-GFP) was microinjected into the cytoplasm of mouse zygotes prior to embryo transfer. Ninety-seven percent (31/32) of the adult mice were confirmed transgenic by PCR and Southern blot analysis; all founder mice express GFP when tail snips were examined by fluorescent microscopy prior to genomic DNA extraction. Transgene insertion numbers ranging from 1 to 32 were revealed by Southern blot analysis. Germline transmission was confirmed by the presence of transgene in F1 offspring. As expected, a lower transgenic rate (2.2%; 1/46) resulted when envelope-free LVU-GFP was microinjected into the perivitelline space (PVS) because cell recognition followed by membrane fusion between the viral envelope and the target cell is prerequisite for successful infection by envelope viruses. Here we demonstrate the competence of envelope-free lentivirus in establishing stable gene integration by germline transgenesis in mice at high efficiency, by intracytoplasmic viral injection (INVI) of envelope-free lentivirus into mouse zygotes.     

No evidence for germ-line transmission following prenatal and early postnatal AAV-mediated gene delivery

BACKGROUND: Recombinant adeno-associated viruses have been used successfully in a number of pre-clinical and clinical gene therapy studies. Since there is a broad consensus that gene therapy must not lead to germ-line transmission, the potential of such vectors for inadvertent gene transfer into germ cells deserves special attention. This applies in particular to pre- or perinatal vector application which has been considered for diseases presenting with morbidity already at birth. METHODS: AAV serotype 2 derived vectors carrying a beta-galactosidase reporter gene or human clotting factor IX cDNA were injected intraperitoneally or via a yolk sac vein into mouse fetuses or administered intravascularly to newborn mice. Tissue samples of the treated animals including the gonads as well as sperm DNA, obtained by differential lysis of one testis of each male animal, and the offspring of all treated mice were investigated for the presence of vector DNA by nested PCR. In positive samples, the copy number of the vector was determined by quantitative real-time PCR. RESULTS: AAV vectors administered intraperitoneally or intravascularly to fetal or newborn mice reached the gonads of these animals and persisted there for time periods greater than one year. Intravascular injection of the vector resulted more frequently in gene transfer to the gonads than intraperitoneal injection. Vector copy numbers in the gonads ranged from 0.3 to 74 per 10(4) cell equivalents. However, neither in isolated sperm DNA from the treated animals nor in their offspring were vector sequences detectable. CONCLUSIONS: These data suggest the risk of inadvertent germ-line transmission following prenatal or early postnatal AAV type 2 mediated gene delivery to be very low.     

Establishment of a rapid and scalable gene expression system in livestock by site-specific integration

Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site.     

A Method for Uptake Quantification of Multiple Fluorescent DNAs in Boar Semen As an Alternative to Radiolabeling

Sperm-mediated gene transfer (SMGT) is a simple and efficient method for producing multitransgenic organisms. Until now, the exogenous DNA uptake efficiencies have been quantified, performing coincubation of spermatozoa with ³H-DNA. This method has significant limitations; from a researcher''s point of view, radioactivity-based experiments are hazardous and require specialistic skills, and in technical analysis, the signal does not allow the simultaneous discrimination of two or more types of labeled constructs. Considering these remarkable points, the present work aims to develop a method for differential uptake quantification of various transgenes alternative to the use radioactive material. The main approach relies on fluorescent-specific peaks for each construct, and their diminution during the sperm—DNA-coincubation phase. The obtained results were confirmed by real-time PCR analysis and fluorescence microscopy imaging. This method becomes of primary importance when the SMGT technique has to be applied on various constructs, as it allows preliminary conclusions to be drawn about multiple transgenesis events and to approach further research about eventual sperm membrane preferences in sequences or structures for constructs.     

应用肌肉介导基因转移制备5,10亚甲基四氢叶酸还原酶(MTHFR)抗体

５,１０亚甲基四氢叶酸还原酶（ＭＴＨＦＲ）是叶酸代谢的关键酶．为了试验肌肉介导外源基因人ＭＴＨＦＲ（ｈＭＴＨＦＲ）制备抗体和建立ＭＴＨＦＲ免疫检测的可能性,构建了ＭＴＨＦＲ基因真核表达载体（ｐｃＤＮＡ３／ＭＴＨＦＲ）;通过基因缝线法将携带ｐｃＤＮＡ３／ＭＴＨＦＲ的质粒,缝合于预先注射再生剂（丁哌卡因）的肌肉内．２个月后分离血清,所得抗体应用Ｗｅｓｔｅｒｎｂｌｏｔ,ＥＬＩＳＡ和胎肝免疫组织化学染色进行免疫鉴定．胎肝免疫组织化学显示,在肝小梁细胞浆中具有大量ＭＴＨＦＲ阳性反应颗粒;Ｗｅｓｔｅｒｎｂｌｏｔ有ＭＴＨＦＲ抗体与其抗原特异的褐色条带,分子量约为３７ｋＤ;ＥＬＩＳＡ分析表明,３种不同浓度的抗体与不同剂量的抗原反应具有剂效关系,最适抗体滴度（ＥＤ５０）为１∶４００。以上结果说明肌肉介导外源基因是获得抗体的一种简单、快捷的方法．该抗体可用于ＭＴＨＦＲ的免疫检测和有关的叶酸代谢研究工作．     

Using a modified piggyBac transposon-combined Cre/loxP system to produce selectable reporter-free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer

Transposon systems are widely used for genetic engineering in various model organisms. PiggyBac (PB) has recently been confirmed to have highly efficient transposition in the mouse germ line and mammalian cell lines. In this study, we used a modified PB transposon system mediated by PB transposase (PBase) mRNA carrying the human lactoferrin gene driven by bovine β-casein promoter to transfect bovine mammary epithelial cells (BMECs), and the selectable reporter in two stable transgenic BMEC clones was removed using cell-permeant Cre recombinase. These reporter-free transgenic BMECs were used as donor cells for somatic cell nuclear transfer (SCNT) and exhibited a competence of SCNT embryos similar to stable transgenic BMECs and nontransgenic BMECs. The comprehensive information from this study provided a modified approach using an altered PB transposon system mediated by PBase mRNA in vitro and combined with the Cre/loxP system to produce transgenic and selectable reporter-free donor nuclei for SCNT. Consequently, the production of safe bovine mammary bioreactors can be promoted.     

Retroviral-mediated gene transfer in primary murine and human T-lymphocytes

Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures that the foreign genetic material will be faithfully transmitted to the cells’ progeny. However, oncoretroviral vectors derived from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral modiated gene transfer efficiency in primary T-lymphocytes, in particular T cell activation status, viral receptor expression, and culture conditions.     

Sperm nuclear transfer and transgenic production in the fish medaka

Sperm nuclear transfer or intracytoplasmic sperm injection (ICSI) is a powerful assisted reproductive technology (ART) for treating human male infertility. Controversial reports of increased birth defects have raised concerns about the ART's safety. The cause for birth defects, however, has remained elusive for analysis in human because of the sample size, male infertility genetics, physiological heterogeneity and associated procedures such as embryo manipulations. Animal models are required to evaluate factors leading to the increased birth defects. Here we report the establishment of medakafish model for ICSI and transgenic production. This small laboratory fish has high fecundity and easy embryology. We show that ICSI produced a 5% high percentage of fertile animals that exhibited both paternal and maternal contribution as evidenced by the pigmentation marker. Furthermore, when sperm were pre-incubated with a plasmid ubiquitously expressing RFP and subjected to ICSI, 50% of sperm nuclear transplants showed germline transmission. We conclude that medaka is an excellent model for ICSI to evaluate birth defects and that sperm nuclear transfer can mediate stable gene transfer at high efficiency. Although more demanding for experimentation, sperm-mediated transgenesis should be particularly applicable for aquaculture species with a lengthy generation time and/or a large adult body size.     

DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HJV)-liposomes

Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor.     

利用雄性生殖细胞建立转基因动物

追溯了用雄性生殖细胞建立转基因动物的发展历程 ,系统阐述了本领域理论和实践的最新进展 ,围绕方法学逐渐改进和完善的过程 ,从利用精子和精原干细胞携带外源DNA两个方向展开 ,分析和评价了DNA转移方法与精子载体法结合、胞浆内单精子注射、蛋白连接的精子介导的基因转移、输精管注射法以及曲细精管显微注射法和精原干细胞移植法 6种实验设计方法。