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.     

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.     

Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis

In the present study, we examined the amphibian Xenopus laevis as a model for stable transgenesis and in particular targeted transgene protein expression to the melanotrope cells in the intermediate pituitary. For this purpose, we have fused a Xenopus proopiomelanocortin (POMC) gene promoter fragment to the gene encoding the reporter green fluorescent protein (GFP). The transgene was integrated into the Xenopus genome as short concatemers at one to six different integration sites and at a total of one to approximately 20 copies. During early development the POMC gene promoter fragment gave rise to GFP expression in the total prosencephalon, whereas during further development expression became more restricted. In free-swimming stage 40 embryos, GFP was found to be primarily expressed in the melanotrope cells of the intermediate pituitary. Immunohistochemical analysis of cryosections of brains/pituitaries from juvenile transgenic frogs revealed the nearly exclusive expression of GFP in the intermediate pituitary. Metabolic labelling of intermediate and anterior pituitaries showed newly synthesized GFP protein to be indeed primarily expressed in the intermediate pituitary cells. Hence, stable Xenopus transgenesis with the POMC gene promoter is a powerful tool to study the physiological role of proteins in a well-defined neuroendocrine system and close to the in vivo situation.     

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。     

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual‐labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N‐terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin‐C promoter. mCherry is expressed in almost all tissues analyzed including pre‐ and post‐implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals. genesis 48:723–729, 2010. © 2010 Wiley‐Liss, Inc.     

小鼠ALB启动子/增强子驱动HSV-tk 对肝脏细胞的杀伤效应

利用小鼠白蛋白(ALB)启动子／增强子及单纯疱疹病毒胸苷嘧啶激酶(HSV-tk)DNA构建了载体pLLTK,以研究该载体对肝脏细胞的特异性杀伤效应。首先,为了比较载体的肝脏细胞特异转录活性,以绿色荧光蛋白(GFP)基因为报告基因构建了载体pLE(仅含小鼠ALB启动子)、pLLE(含小鼠ALB启动子和上游增强子)和pLEL(含小鼠ALB启动子和下游增强子),分别转染到人肝细胞株Hep—G2与小鼠乳腺上皮细胞株HC-11,荧光显微镜与流式细胞术分析GFP的表达。然后将载体pLLTK转染到Hep-G2研究对细胞的杀伤效应。结果发现：小鼠ALB启动子／增强子能驱动GFP肝脏特异表达;HSV-tk在Hep-G2表达使细胞具有更昔洛韦(GCV)敏感性,在GCV作用7d后,MTT分析细胞的生存率,pLLTK转染细胞表现明显的细胞死亡(53％),而阴性对照组pcDNA3．1转染细胞没有明显变化(仅2％细胞死亡)。以上结果表明所有的载体具有肝脏细胞特异性,为利用该载体产生肝脏损伤的转基因小鼠提供了细胞水平的实验依据。     

A cyp19a1b‐gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens. genesis 47:67–73, 2009. © 2008 Wiley‐Liss, Inc.     

The intron 5/6 promoter region of the ship1 gene regulates expression in stem/progenitor cells of the mouse embryo

The s-SHIP protein is a shorter isoform of the longer SHIP1 protein and lacks the N-terminal SH2 domain region contained in SHIP1. s-SHIP is expressed in ES cells and in enriched bone marrow stem cells, and may be controlled by a promoter within intron 5 of the ship1 gene. We therefore examined the potential specificity of promoter activity in ES cells of an intron 5/intron 6 ship1 genomic segment and its tissue specificity within transgenic mice expressing GFP from this promoter region. The results indicate that s-SHIP promoter activity is specific for ES cells in vitro and for known and presumptive stem/progenitor cells throughout embryo development of the transgenic mice. Specific GFP expression was observed in the blastocyst, primordial germ cells, thymus, arterioles, osteoblasts, and skin epidermis. The epidermis/epithelium is the progenitor for hair follicles, mammary tissue, and prostate. Interestingly, each of these latter tissues acquired a few GFP-positive cells in the course of their development from the epithelial layers, and these cells express marker proteins for stem/progenitor cells. These results identify potential stem cell populations, mark these cells for analyses in normal and cancer development, and implicate s-SHIP as an important protein in stem/progenitor cell function.     

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter

The fat-1 gene was isolated from roundworm Caenorhabditis elegans, and built into pIRES2-EGFP expression vectors driven by cytomegalovirus (CMV) promoter or cytomegalovirus enhancer and chickenβ-actin (CAG) promoter. Both CMV- and CAG-driven expression vectors were transfected to sheep fetal fibroblast cells. Positive transfected cells were used as donors for somatic cell nuclear transfer (SCNT) and the cloned embryos were transferred into the oviducts of synchronized recipient sheep. Two lambs derived from CMV vector and three lambs derived from CAG vector developed to term. Although Southern analyses using tissues from the two lambs derived from CMV vectors indicated integration of fat-1 gene into the genome, fat-1 mRNAs were not detected by RT-PCR. However, there was fat-1 expression (detected by RT-PCR) in tissues from transgenic lambs driven by CAG vectors. To investigate potential mechanisms involved in the two transgene models, methylation state of the vector promoters were examined. In CMV-driven transgenics, CMV promoters had almost no methylation in transfected cells and the resultant cloned embryos, whereas high methylations were detected in tissues and organs in transgenic lambs. In the CAG-driven transgenics, there were almost no methylations in transgenic cells and transgenic cloned embryos, and cloned lambs expressed fat-1 mRNA (detected by RT-PCR). Moreover, although SV40 promoters which drove neo/kan marker gene in CMV vectors were highly methylated in tissues from transgenic lambs, they were without methylation in cells and embryos. Therefore, we concluded that highly methylated CMV promoters induced the silence of fat-1 transgene expression in sheep. Furthermore, CAG promoter, but not CMV promoter was suitable for generation of fat-1 transgenic sheep.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest‐derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest‐derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell‐type‐specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin‐synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.     

用绿色荧光蛋白监测转基因植物中选择标记基因的消除

绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。     

In vivo gene transfer to mouse spermatogenic cells using green fluorescent protein as a marker

Combination of the DNA injection into seminiferous tubules and the subsequent in vivo electroporation (EP) has become an efficient and convenient assay system for spermatogenic-specific gene expression during spermatogenesis of mice. In this study, we made methodological modifications to enhance the transfection efficiency, and evaluated the possibility of this technique to generate transgenic offspring using green fluorescent protein (GFP) as a marker. After the in vivo gene transfer, GFP expression could be monitored easily and repeatedly on the surface of the testis of live mice under fluorescent microscopy. The serial sections of the transfected testis revealed that transient expression of GFP was extended even in the innermost region of the testis uniformly, but confined to spermatogenic cells and Sertoli cells within the seminiferous tubules. Furthermore, long-lasting GFP expression could be detected in the spermatogenic cells even 2 months after EP. Natural mating with normal adult females revealed that 65% of the transfected males maintained fertilizable ability and could generate their offspring normally. Germ-line transmission of the GFP vector to the offspring was checked under fluorescent microscopy, but no transgenic offspring has been detected up to now. These results suggest that the application of additional techniques, such as cell sorting for GFP-positive germ cells followed by nuclear transfer to the oocytes, would make this method as a novel strategy for generating transgenic animals. J. Exp. Zool. 286:212-218, 2000.     

Lentivirus‐mediated bifunctional cell labeling for in vivo melanoma study

Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long‐term culture and colony formation of several LV‐labeled mouse melanoma cells showed that promoters derived from mammalian house‐keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase–green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP‐labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP‐positive cells can be isolated from the tumors by fluorescence‐activated cell sorter. Pol2‐Luc/GFP labeling, while lower in activity, was more sustainable than FerH‐Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol‐2‐Luc/GFP labeling allows long‐term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models.     

Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system

Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.