Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgeulc animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein （EGFP） transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.     

Oviduct-specific enhanced green fluorescent protein expression in transgenic chickens

In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white.     

Establishment of porcine transgenic embryonic germ cell lines expressing enhanced green fluorescent protein

The purpose of this study was to isolate porcine embryonic germ (EG) cells and establish transgenic EG cell lines. Plasmid DNA was the enhanced green fluorescent protein (EGFP) vector. Porcine EG cells in rapid proliferation (4th to 9th passage) were transfected with LipofecTamine 2000 and TransFast reagents. Porcine EG cells transfected with a complex of 1 microg of DNA and 2 microL of LipofecTamine 2000 reagent yielded four EG-EGFP cell lines, which emitted bright green fluorescence. EG-EGFP cells cultured for more than 2 weeks without passage gave rise to various differentiated phenotypes. In addition, to determine the degree to which EG cells become integrated into the inner cell mass of host embryos, 135 embryos were injected with porcine EG-EGFP cells; 110 embryos survived and developed into blastocysts (81.5%). Eighty-four chimeric embryos contained fluorescent cells after culture; 49 blastocysts contained EG-EGFP cells in the inner cell mass. Our results suggested that the chimeric rate would not be improved via using different stages of embryos for injection.     

High level Purkinje cell specific expression of green fluorescent protein in transgenic mice

The green fluorescent protein (GFP) has become a powerful tool in molecular and cell biology. It is a commonly used marker for cloning and transfection experiments as well as a useful label of living cells allowing continuous observation of developing structures. In order to unravel mechanisms of neuronal differentiation, we generated a transgenic mouse model which expresses GFPS65T,hu under the control of the Purkinje cell-specific promoter L7/pcp-2. Here, we show that GFPS65T,hu is highly expressed specifically in the cerebellum in whole mount preparations after the 2nd postnatal week. GFPS65T,hu can be detected exclusively in Purkinje cells of cerebellar slices. The fluorescence intensity of GFPS65T,hu should enable the characterization and recording of axons, dendrites, and spines protruding from these neuronal processes. The level of GFP expression can be quantified by western blotting which allows to analyze protein expression and L7/pcp-2 promoter regulation in vivo. The application of cellular and physiological techniques on L7GFP mice will provide a remarkable opportunity to investigate various aspects of neuronal development at the cellular and subcellular levels.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A + C + 0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A + C + 0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

Enhanced green fluorescent protein as a useful tag for rapid identification of homozygous transgenic mice

We developed a technique that simplifies the process of confirming homozygous transgenics at preimplantation stages, which are the earliest stages used in test breeding, using enhanced green fluorescent protein as a tag. All the blastocysts obtained by mating with the combination of Tg/Tg male (homozygous for transgene) x +/+ female exhibited fluorescence.     

The utility of green fluorescent protein in transgenic plants

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has proven to be a powerful tool in plant genetic transformation studies. This paper reviews the history and the progression of the expression of GFP variants in transgenic plants. The distinguishing features of the most useful GFPs, such as those including the S65T chromophore mutation and those with dual excitation peaks, are discussed. The review also focuses on the utility of GFP as a visual selectable marker in aiding the plant transformation process; GFP has been more important in monocot transformation compared with dicot transformation. Finally, the potential utility of new fluorescent proteins is speculated upon.     

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.     

Assessment of anti-estrogenic activity of tamoxifen in transgenic mice expressing an enhanced green fluorescent protein gene regulated by estrogen response element

Tamoxifen is an anti-estrogenic agent for the treatment of breast cancer, while exhibiting estrogenic activity in such tissues as the uterus. This study aimed to test whether these opposite properties of tamoxifen in the uterus can be evaluated separately in vivo. We employed two transgenic murine models named, respectively, the ERE-EGFP Ar+/+ mouse and ERE-EGFP Ar-/- mouse. Both types of mice possess an enhanced green fluorescent protein (EGFP) gene regulated by four copies of estrogen response elements (EREs), while the latter lacks a functional aromatase gene, which encodes an enzyme catalyzing conversion of androgens to estrogens. Tamoxifen clearly exhibited estrogenic activity in the uteri of ERE-EGFP Ar-/- mice, as it caused uterine wet weight gain and E2-target gene induction, as 17beta-estradiol (E2) did. However, tamoxifen did not enhance the EGFP expression in ERE-EGFP Ar-/- mice, although E2 induced it significantly. In ERE-EGFP Ar+/+ mice, tamoxifen suppressed the EGFP expression in a time- and dose-dependent manner. Thus, the present study demonstrated that estrogenic and anti-estrogenic activities of tamoxifen can be evaluated by using ERE-EGFP Ar-/- and ERE-EGFP Ar+/+ mice, respectively. Furthermore, these animal models are useful to select and evaluate estrogenic and anti-estrogenic activities of chemical compounds.     

Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein

We have generated embryonic stem (ES) cells and transgenic mice carrying a tau-tagged green fluorescent protein (GFP) transgene under the control of a powerful promoter active in all cell types including those of the central nervous system. GFP requires no substrate and can be detected in fixed or living cells so is an attractive genetic marker. Tau-tagged GFP labels subcellular structures, including axons and the mitotic machinery, by binding the GFP to microtubules. This allows cell morphology to be visualized in exquisite detail. We test the application of cells derived from these mice in several types of cell-mixing experiments and demonstrate that the morphology of tau-GFP-expressing cells can be readily visualized after they have integrated into unlabeled host cells or tissues. We anticipate that these ES cells and transgenic mice will prove a novel and powerful tool for a wide variety of applications including the development of neural transplantation technologies in animal models and fundamental research into axon pathfinding mechanisms. A major advantage of the tau-GFP label is that it can be detected in living cells and labeled cells and their processes can be identified and subjected to a variety of manipulations such as electrophysiological cell recording.     

辅助病毒依赖型腺病毒载体转基因的体外表达效率

构建可表达增强型绿色荧光蛋白 (Enhanced green fluorescent protein,EGFP) 的辅助病毒依赖型腺病毒载体 (Helper-dependent adenoviral vector,HDAd),并完成大量制备、纯化和体外表达鉴定。荧光显微镜证实HDAd/EGFP可表达,电镜下观察到经CsCl纯化后的腺病毒的典型形态。分光光度计法测定病毒的浓度为4.0×1012 颗粒数 (Virus particle,vp) /mL。与可表达EGFP的第一代腺病毒载体 (First generation adenoviral vector,FGAd) FGAd/EGFP进行了体外感染和转基因表达效率的比较研究,分别用约2 000 vp/细胞的HDAd/EGFP和FGAd/EGFP感染A549细胞,流式细胞仪检测EGFP的表达情况。通过相同时间点流式细胞仪分析EGFP的表达情况,可见HDAd/EGFP感染早期的A549细胞较FGAd/EGFP有更高的荧光表达率及更高的表达强度,显示HDAd载体具有转基因瞬时高表达的特性,是一种更有价值的疫苗载体。     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A+C+0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A+C+0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

Embryo culture-based generation of enhanced green fluorescent protein-transgenic mice

One of the limitations of transgenesis is low efficiency. In this study, we generated transgenic mice harboring the enhanced green fluorescent protein (EGFP) gene, under the control of chicken-beta-actin promoter and cytomegalovirus enhancer, using two approaches and compared their efficiencies. One involved culture of EGFP-injected embryos developing through EGFP-expressing "green" blastocysts, followed by their transfer to uterus. The second was oviductal-transfer of EGFP-injected-eggs. Embryo culture-based-transgenesis (ECBT) produced 100% transgenic mice, unlike the second approach. Moreover, ECBT required reduced number of recipients and markedly increased pregnancy rates. Of the nine founders, seven exhibited ubiquitous EGFP-expression, one (GU1) was a mosaic and the other (G18) was non-expressing. The molecular basis for this was attributed to repeat-induced gene silencing, since the G18 had a high copy number (approximately 99/genome) of the non-mutated and non-rearranged EGFP-transgene integrated at a single site. Our results show the superiority of ECBT over the conventional oviductal approach for generating transgenic "green" mice.     

Modification of enhanced green fluorescent protein for secretion out of cells

Since its discovery approximately 20 years ago, green fluorescent protein (GFP) has become one of the most widely used reporter proteins. GFP has been used in a variety of living organisms, ranging from E. coli to higher eukaryotes, such as plants and animals. The biggest advantage of using this reporter protein is that it can be used to monitor in vitro and in vivo gene expression. One important limitation, however, is its inability to be secreted out of cells. For this reason, it has been difficult to directly measure the expression level of the regulatory sequence of a gene of interest quantitatively. To overcome this drawback, we have modified the enhanced green fluorescent protein gene (EGFP), a derivative of GFP, by adding a signal peptide sequence that encodes a rat follicle-stimulating hormone (FSH) β-subunit upstream of EGFP. Following the expression of this modified gene in several cell types, we have found efficient secretion of EGFP. Consequently, with the secreted protein, we could easily quantify the gene expression level with high reliability. Therefore, the use of our modified EGFP expression cassette would greatly facilitate the evaluation of regulatory sequences, such as promoters and enhancers. Further, it will also be very helpful in the study of transgenic livestock intended to use as bioreactors for mass production of pharmaceuticals.     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。     

Laser Scanning Cytometry for selection of green fluorescent protein transgenic mice using small number of blood cells

A Laser Scanning Cytometry-based method was developed for identification of transgenic mice expressing green fluorescent protein (GFP) using minute amounts of peripheral blood. The difference between the autofluorescence of cells not expressing GFP and the fluorescence of GFP expressing cells after excitation with Ar-ion laser (wavelength 488 nm) and detection of emitted fluorescent light in the green channel was high enough for unambiguous identification of the GFP expressing mice. The sensitivity of this method was estimated 1:10(4) for detection of rare GFP expressing cells under the conditions used. This sensitivity should be sufficient for many studies on microchimerism. Because of the possibility for relocation of the cells, this method will be particularly useful for characterizing the cells with high GFP expression using other markers of cell phenotype or conventional morphological analysis.     

A desolvation model for trifluoroethanol-induced aggregation of enhanced green fluorescent protein

Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of β-sheet-rich, flexible fibrils for acid-denatured EGFP. By fitting our circular dichroism data to a model in which TFE-water mixtures are assumed to be ideal solutions, we show that increasing entropic costs of protein solvation in TFE-water mixtures may both cause the population of the intermediate state and increase aggregate production. Tertiary structure and electrostatic repulsion also impede aggregation. We conclude that initiation of EGFP aggregation in TFE likely involves overcoming of multiple protective factors, rather than stabilization of aggregation-prone structural elements.