Cre recombinase expression in cerebellar Purkinje cells

The cerebellar cortex and its sole output, the Purkinje cell, have been implicated in motor coordination, learning and cognitive functions. Therefore, the ability to generate Purkinje cell-specific mutations in physiologically relevant genes is of particular neurobiological interest. A suitable approach is the Cre/loxP strategy that allows temporally and spatially controlled gene inactivation. Here, we present the characterization of transgenic mouse strains expressing Cre recombinase controlled by the L7/pcp-2 gene. Endogenous L7/pcp-2 protein is expressed exclusively in Purkinje cells and retinal bipolar neurones. Recombination was detected by beta-galactosidase histochemistry in tissues from crosses of the L7/pcp-2:Cre transgenic lines with two different indicator strains, GtROSA26 and ACZL. Purkinje cells in all folia of the cerebellum displayed intense beta-galactosidase staining, whereas only few blue cells were observed in the retina and other parts of the CNS. Thus, these transgenic lines are potentially of great importance for genetic manipulations in cerebellar Purkinje cells.     

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.     

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.     

Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions.

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the cre gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus. In addition, the GFP cre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.     

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.     

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.     

The Construction of nifH-gfp Expression Vector and Its Expression in Enterobacter gergoviae 57-7

A nifH-gfp expression vector pMGFP2 was constructed by fusing the 725 bp PCR amplified triple mutated green fluorescent protein (GFP) gene (gfpS65T,V68L,S72A) fragment to the nifH promoter and its start codon which was from Klebsiella pneumoniae (Schr eter) Trevisan M5a1. A kanamycin cassette was inserted into PstⅠ site of pMGFP2, obtaining the expressing vector pMGFP2.1 which can be used for the studying of nifH-gfp expression in Enterobacter gergoviae 57-7. It was then transformed into E.gergoviae 57-7 and the effects of NH+4 and oxygen on the expression of nifH-gfp in E. gergoviae 57-7 were studied.     

Molecular regulation of MHC class I chain-related protein A expression after HDAC-inhibitor treatment of Jurkat T cells

In this study, we characterize the molecular signal pathways that lead to MHC class I chain-related protein A (MICA) expression after histone deacetylase (HDAC)-inhibitor (HDAC-i) treatment of Jurkat T cells. Chelating calcium with BAPTA-AM or EGTA potently inhibited HDAC- and CMV-mediated MICA/B expression. It was further observed that endoplasmic reticulum calcium stores were depleted after HDAC treatment. NF-kappaB activity can be induced by HDAC treatment. However, nuclear translocation of NF-kappaB p65 was not observed after HDAC treatment of Jurkat T cells and even though we could effectively inhibit p65 expression by siRNA, it did not modify MICA/B expression. To identify important elements in MICA regulation, we made a promoter construct consisting of approximately 3 kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a germinal center-box containing a putative Sp1 site from position -113 to -93 relative to the mRNA start site was important for HDAC and CMV-induced promoter activity. Sp1 was subsequently shown to be important, as targeted mutation of the Sp1 binding sequence or siRNA mediated down modulation of Sp1-inhibited MICA promoter activity and surface-expression.     

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.     

Real-time flow cytometric quantification of GFP expression and Gfp-fluorescence generation in Saccharomyces cerevisiae

A genetic and analytical methodology was developed based on a green fluorescent mutant protein (Gfp(S65T)) that allows the real-time quantification of gene expression in Saccharomyces cerevisiae. Using the UAS(GAL)(1-10)/CYC1 promoter and plasmids that are maintained in different copy numbers per cell, wild-type GFP and mutant GFP(S65T) were expressed in low to high concentration. Flow cytometric analysis was then applied to directly quantify Gfp((S65T)) (both wild type and mutant protein) expression at the single-cell level, and to indirectly measure the concentrations of non-fluorescent apoGfp((S65T)) and fluorescent Gfp((S65T)), which is autocatalytically formed from the apoprotein. Kinetics of apoGfp((S65T))/Gfp((S65T)) conversion during aerobic growth showed that the time required for complete apoGfp((S65T)) conversion is limited only by the amount of apoprotein that is expressed. When GFP(S65T) was expressed in single copy, the apoprotein did not accumulate and was instantly converted into its fluorescent form. The data indicate that an instant quantification of gene expression in S. cerevisiae is achievable based on Gfp(S65T), even if the gene is transcribed from a very strong promoter.     

A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation.

Green fluorescent protein (GFP) has been utilized as a powerful reporter of gene expression and protein localization in cells. We discovered a mutant carrying point mutation S208L from a UV-excitable GFP (F99S/M153T/V163A). It had the enhanced fluorescence intensity. Introduction of the red-shifted mutations (F64L/S65T) to this mutant led to the GFP having the brightest mutants reported which were expressed in Escherichia coli and excited at 488 nm. The relative fluorescence intensities to that of wild-type GFP and GFPuv were increased about 120- and 10-fold, respectively. It was shown that the S208L mutation contributes to both a higher intrinsic brightness of GFP and a higher expression level in E. coli.     

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.     

Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43

Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of connexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomatitis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-express the green fluorescent protein (GFP) and Cx43 in the communication-deficient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx43.GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 x 10(7) colony form units (CFU)/ml and was free of replication-competent retroviruses. After a 7-day treatment with retinoic acid (20 microm), N2a transformants (N2a-Cx43 and N2a-Cx43.GFP) maintained the expression of Cx43 and Cx43.GFP. Expression of both constructs resulted in functional coupling, as evidenced by electrophysiological and dye-injection analysis. Suppression of cell growth correlated with expression of both Cx43 or Cx43.GFP and retinoic acid treatment. Based on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expressing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2a cells does not alter retinoic acid-induced neuronal differentiation but does enhance growth inhibition.     

Combinatorial marking of cells and organelles with reconstituted fluorescent proteins

Expression of GFP and other fluorescent proteins depends on cis-regulatory elements. Because these elements rarely direct expression to specific cell types, GFP production cannot always be sufficiently limited. Here we show that reconstitution of GFP, YFP, and CFP previously split into two polypeptides yields fluorescent products when coexpressed in C. elegans. Because this reconstitution involves two components, it can confirm cellular coexpression and identify cells expressing a previously uncharacterized promoter. By choosing promoters whose expression patterns overlap for a single cell type, we can produce animals with fluorescence only in those cells. Furthermore, when one partial GFP polypeptide is fused with a subcellularly localized protein or peptide, this restricted expression leads to the fluorescent marking of cellular components in a subset of cells.     

Is green fluorescent protein toxic to the living cells?

Green fluorescent protein (GFP) has become more popular to be used as a living marker for positively transfected clones in many studies. To establish stable cell lines constitutively expressing GFP, three GFPs expressed from plasmid pBIEGFP, pSG5GFP, and pRSGFP were introduced into NIH/3T3, BHK-21, Huh-7, and HepG2 cells. All the GFPs we used are the mutant forms of a common wild phenotype. The pBIEGFP expressed enhanced GFP (EGFP). The pRSGFP and pSG5GFP expressed red shift GFP (RSGFP). The RSGFP gene in pSG5GFP was driven by a strong SV40 promoter and showed at least 20-fold higher RSGFP expression by western blot analysis. Despite of the variation in the levels of GFP expression, many GFP expressing cells contracted, rounded-up, and died, which was confirmed by decreasing luciferase activity. CPP32 activity and flow cytometric analyses further demonstrate that cells expressing GFP underwent apoptosis. Our observation is contradictory to other reports that GFP is nontoxic to the cells. Most importantly, this paper shows for the first time the link between expression of GFP and induction of apoptosis. This finding should promote studies of GFP cytotoxicity and attempts to isolate new non-toxic mutants of GFP.     

Optimization of Single-Cell Electroporation Protocol for Forced Gene Expression in Primary Neuronal Cultures

The development and function of the central nervous system (CNS) are realized through interactions between many neurons. To investigate cellular and molecular mechanisms of the development and function of the CNS, it is thus crucial to be able to manipulate the gene expression of single neurons in a complex cell population. We recently developed a technique for gene silencing by introducing small interfering RNA into single neurons in primary CNS cultures using single-cell electroporation. However, we had not succeeded in forced gene expression by introducing expression plasmids using single-cell electroporation. In the present study, we optimized the experimental conditions to enable the forced expression of green fluorescent protein (GFP) in cultured cerebellar Purkinje neurons using single-cell electroporation. We succeeded in strong GFP expression in Purkinje neurons by increasing the inside diameter of micropipettes or by making the size of the original plasmid smaller by digestion and cyclizing it by ligation. Strong GFP expression in Purkinje neurons electroporated under the optimal conditions continued to be observed for more than 25 days after electroporation. Thus, this technique could be used for forced gene expression in single neurons to investigate cellular and molecular mechanisms of the development, function, and disease of the CNS.     

A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene

To search for genes involved in determining the morphology of individual neuronal types, a cDNA library was constructed from postnatal day 13 mouse cerebellum. From this library, 2 clones, L7 and L19, were isolated by a differential hybridization procedure and shown by in situ hybridization to be Purkinje cell-specific within the cerebellum. Both RNAs appear between postnatal days 4 and 8 and continue into adulthood, coinciding with terminal differentiation of the Purkinje cells. L7 seems to be expressed exclusively in the cerebellum, whereas L19 is expressed throughout the brain. Consistent with the RNA localization, L7 protein is found only in the cerebellum and is confined to the Purkinje cells. The L7 amino acid sequence has been deduced from the cDNA sequence, and a pseudo-repeat within the L7 protein sequence is homologous to the amino acids sequence in the primary translation product of the gene for human sis/PDGF.