Dual promoter expression system with insulator ensures a stringent tissue-specific regulation of two reporter genes in the transgenic fish

The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5′-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.     

Expression of GFP in nuclear transplants generated by transplantation of embryonic cell nuclei from GFP-transgenic fish into nonenucleated eggs of medaka, Oryzias latipes

In order to investigate whether foreign genes can be used as genetic markers of donor nuclei in fish nuclear transplantation, expression of the GFP gene derived from donor nuclei was examined in nuclear transplants in medaka (Oryzias latipes). Embryonic nuclei were obtained from blastula embryos produced by crossing of transgenic fish of the wild-type strain heterozygous for the GFP gene with nontransgenic ones or by mutual crossing between transgenic fish. The GFP gene was driven by the promoter of the medaka elongation factor gene, EF-1alpha-A, which is known to induce GFP expression in many tissues except for the muscle in the transgenic fish. The nuclei were transplanted into nonenucleated unfertilized eggs of the orange-red strain. Adult nuclear transplants were successfully obtained at the rate of about 2% of the operated eggs. They were triploid and had no reproductive potential. The GFP gene was expressed in embryos, fry, and adults of nuclear transplants in a pattern similar to that in the transgenic fish. These results indicate that GFP is useful as a foreign genetic marker of donor nuclei in fish nuclear transplantation.     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules

We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.Communicated by W. Harwood     

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.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1α enhancer-promoter (EF1). Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system, were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression in living cells is useful for characterizing the activity of cis-elements in vivo. Received December 21, 1998; accepted March 31, 1999.     

Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum

We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression.     

Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos

A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys.     

Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot meristematic cultures

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic oat ( Avena sativa L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by a rice actin promoter. In vitro shoot meristematic cultures (SMCs) induced from shoot apices of germinating mature seeds of a commercial oat cultivar, Garry, were used as a transformation target. Proliferating SMCs were bombarded with a mixture of plasmids containing the sgfp(S65T) gene and one of three selectable marker genes, phosphinothricin acetyltransferase (bar), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII). Cultures were selected with bialaphos, hygromycin B and geneticin (G418), respectively, to identify transgenic tissues. From 289 individual explants bombarded with the sgfp(S65T) gene and one of the three selectable marker genes, 23 independent transgenic events were obtained, giving a 8.0% transformation frequency. All 23 transgenic events were regenerable, and 64% produced fertile plants. Strong GFP expression driven by the rice actin promoter was observed in a variety of tissues of the T(0) plants and their progeny in 13 out of 23 independent transgenic lines. Stable GFP expression was observed in T(2) progeny from five independent GFP-expressing lines tested, and homozygous plants from two lines were obtained. Transgene silencing was observed in T(0) plants and their progeny of some transgenic lines.     

Green fluorescent protein as a marker in transgenic mice

Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.     

Visualization of rod photoreceptor development using GFP-transgenic zebrafish

Zebrafish retina contains five morphologically distinct classes of photoreceptors, each expressing a distinct type of opsin gene. Molecular mechanisms underlying specification of opsin expression and differentiation among the cell types are largely unknown. This is partly because mutants affected with expression of a particular class of opsin gene are difficult to find. In this study we established the transgenic lines of zebrafish carrying green fluorescent protein (GFP) gene under the 1.1-kb and 3.7-kb upstream regions of the rod-opsin gene. In transgenic fish, GFP expression initiated and proceeded in the same spatiotemporal pattern with rod-opsin gene. The retinal section from adult transgenic fish showed GFP expression throughout the rod cell layer. These results indicate that the proximal 1.1-kb region is sufficient to drive gene expression in all rod photoreceptor cells. These transgenic fish should facilitate screening of mutants affected specifically with rod-opsin expression or rod cell development by visualization of rod cells by GFP.     

Fluorescence visualization of ultraviolet-sensitive cone photoreceptor development in living zebrafish

Cone photoreceptor cells of fish retinae are arranged in a highly organized fashion. However, the molecular mechanisms underlying photoreceptor development and retinal pattern formation are largely unknown. Here we established transgenic lines of zebrafish carrying green fluorescent protein (GFP) cDNA with the 5.5-kb upstream region of the ultraviolet-sensitive cone opsin gene (SWS1). In the transgenic fish, GFP gene expression proceeded in the same spatiotemporal pattern as SWS1 in the retinae of embryos. In the adult retina, GFP expression was observed throughout the short single cone (SSC) layer where SWS1 is specifically expressed. Therefore, the transgenic fish provides an excellent genetic background to study retinal pattern formation, photoreceptor determination and differentiation, and factors regulating these processes and SSC-specific expression of SWS1.     

Preimplantation screening for transgenesis using an embryonic specific promoter and green fluorescent protein

We report enrichment in the efficiency of generating mice transgenic for expression of a human protein in their milk using GFP-mediated preimplantation screening. The transgene array consisted of a functional gene (human alpha-1 antitrypsin under the control of the ovine BLG promoter) linked 5' to a reporter gene (GFP under the control of the murine Oct-4 promoter). GFP expression was detected in blastocysts by fluorescence microscopy and green and nongreen embryos were transferred to recipients in separate groups. In the first experiment, of seven pups that resulted from the transfer of blastocysts expressing GFP, five (71%) were transgenic. The experiment was repeated and of 12 pups that resulted from transfer of GFP-expressing blastocysts, 11 were transgenic (92%). The presence of the reporter cassette used for preimplantation screening did not affect the expression level of alpha-1-antitrypsin in the milk of the transgenic mice. In addition, in a related experiment wherein the GFP reporter gene was co-injected with a second mammary-specific transgene, pINC, no effect on transgene expression was observed. For mice transgenic for the mammary-specific gene alone, expression levels for four different lines were 192, 197, 382, and 415 microg/mL. For mice transgenic for both the mammary-specific transgene and the Oct4-GFP reporter cassette, expression levels for seven different lines were 282, 321, 468, 497, 499, 516, and 806 microg/mL.     

The Murine Stem Cell Virus Promoter Drives Correlated Transgene Expression in the Leukocytes and Cerebellar Purkinje Cells of Transgenic Mice

The murine stem cell virus (MSCV) promoter exhibits activity in mouse hematopoietic cells and embryonic stem cells. We generated transgenic mice that expressed enhanced green fluorescent protein (GFP) under the control of the MSCV promoter. We obtained 12 transgenic founder mice through 2 independent experiments and found that the bodies of 9 of the founder neonates emitted different levels of GFP fluorescence. Flow cytometric analysis of circulating leukocytes revealed that the frequency of GFP-labeled leukocytes among white blood cells ranged from 1.6% to 47.5% across the 12 transgenic mice. The bodies of 9 founder transgenic mice showed various levels of GFP expression. GFP fluorescence was consistently observed in the cerebellum, with faint or almost no fluorescence in other brain regions. In the cerebellum, 10 founders exhibited GFP expression in Purkinje cells at frequencies of 3% to 76%. Of these, 4 mice showed Purkinje cell-specific expression, while 4 and 2 mice expressed GFP in the Bergmann glia and endothelial cells, respectively. The intensity of the GFP fluorescence in the body was relative to the proportion of GFP-positive leukocytes. Moreover, the frequency of the GFP-expressing leukocytes was significantly correlated with the frequency of GFP-expressing Purkinje cells. These results suggest that the MSCV promoter is useful for preferentially expressing a transgene in Purkinje cells. In addition, the proportion of transduced leukocytes in the peripheral circulation reflects the expression level of the transgene in Purkinje cells, which can be used as a way to monitor transgene expression properties in the cerebellum without invasive techniques.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

Neogenesis of cerebellar Purkinje neurons from gene-marked bone marrow cells in vivo.

The versatility of stem cells has only recently been fully recognized. There is evidence that upon adoptive bone marrow (BM) transplantation (BMT), donor-derived cells can give rise to neuronal phenotypes in the brains of recipient mice. Yet only few cells with the characteristic shape of neurons were detected 1-6 mo post-BMT using transgenic or newborn mutant mice. To evaluate the potential of BM to generate mature neurons in adult C57BL/6 mice, we transferred the enhanced green fluorescent protein (GFP) gene into BM cells using a murine stem cell virus-based retroviral vector. Stable and high level long-term GFP expression was observed in mice transplanted with the transduced BM. Engraftment of GFP-expressing cells in the brain was monitored by intravital microscopy. In a long-term follow up of 15 mo post-BMT, fully developed Purkinje neurons were found to express GFP in both cerebellar hemispheres and in all chimeric mice. GFP-positive Purkinje cells were also detected in BM chimeras from transgenic mice that ubiquitously express GFP. Based on morphologic criteria and the expression of glutamic acid decarboxylase, the newly generated Purkinje cells were functional.     

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.