Green Fluorescent Protein Signal Research Articles

Rac Recruits Aux/IAA Proteins to Deadly Nuclear Bodies

Auxin stimulates gene expression by triggering the proteasome-mediated proteolysis of a family of transcriptional repressors, auxin/indole acetic acid (Aux/IAA) proteins. Tao et al. showed that in transfected protoplasts expressing constitutively active or dominant-negative forms of the guanosine triphosphatase (GTPase) Rac, the activity of Rac controlled the degradation of luciferase-Aux/IAA fusion proteins (Luc-Aux/IAA). Constitutively active Rac stimulated degradation even in the absence of auxin. Furthermore, transfection of GTPase-activating proteins (GAPs) inhibited auxin-stimulated degradation of Luc-Aux/IAA. In protoplasts transfected with green fluorescent protein (GFP)-Aux/IAA fusion proteins under the control of an inducible promoter, the GFP signal was predominantly nuclear, and addition of auxin or coexpression of constitutively active Rac accelerated the rate at which the GFP signal became undetectable following cessation of expression. Dominant-negative Rac stabilized GFP-Aux/IAA. In protoplasts and in roots from seedling plants, auxin treatment triggered the redistribution of the GFP-Auz/IAA proteins from a diffuse nuclear signal to one associated with subnuclear puncta called nuclear protein bodies (NPBs). Inhibition of the proteasome with MG132 or expression of dominant-negative Rac prevented the auxin-induced redistribution of GFP-Aux/IAA to NPBs. Although auxin initially promoted the accumulation of GFP-Aux/IAA into NPBs, ultimately the GFP signal became undetectable, suggesting that these were degradative macromolecular structures. Consistent with this degradative role, auxin also stimulated the accumulation of constituents of the SCF E3 ligases, the COP9 signalosome, and the proteasome in the NPBs when these were coexpressed with a labeled Aux/IAA protein. These results together suggest that auxin acts through Rac to trigger the localization of Aux/IAA proteins into NPBs, which serve as nuclear sites of degradation. L.-z. Tao, A. Y. Cheung, C. Nibau, H.-m. Wu, RAC GTPases in tobacco and arabidopsis mediate auxin-induced formation of proteolytically active nuclear protein bodies that contain AUX/IAA proteins. Plant Cell 17 , 2369-2383 (2005). [Abstract] [Full Text]

Analysis of Green Fluorescent Protein Expression in Transgenic Rats for Tracking Transplanted Neural Stem/Progenitor Cells

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents--Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]--by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wild-type spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.

Syngeneic lymph-node-targeting model of green fluorescent protein-expressing Lewis lung carcinoma

The Lewis lung tumor has been extensively studied in both syngeneic and allogeneic mouse models. However, its metastatic potential and mechanism are poorly understood. The aim of the present study was to develop a highly metastatic lymph-node targeting, imageable model of the Lewis lung carcinoma in a syngeneic host. We report here a syngeneic model of the Lewis lung carcinoma in which the carcinoma cells are labeled with green fluorescent protein (GFP). The tumor cells were transplanted in the dorsal side of the ear of C57-B16 mice in order to give the tumor cells access to the lymphatic system. This model of the Lewis lung carcinoma extensively metastasized to numerous lymph nodes throughout the body of the animal as well as visceral organs, as visualized by fluorescence microscopy using the bright GFP signal. Twenty-one different metastatic sites, including lymph nodes throughout the body, were identified among the cohort of transplanted animals. The data demonstrate a predilection of the Lewis lung carcinoma for lymphatic pathways for metastasis throughout the animal body. The concomitant macrometastases to the visceral organs observed in this model may be remetastasis from the lymph nodes. This model of the Lewis lung carcinoma should be very useful in defining cellular trafficking and targeting mechanisms of metastasis, in particular those involving lymphatic pathways.

In vivo gene transfer to the rat retina using herpes simplex virus type 1 (HSV-1)-based amplicon vectors

The purpose of our study was to evaluate the transduction profiles of herpes simplex virus type 1 (HSV-1)-based amplicon vectors following subretinal injection in the rat. Two amplicon vectors were tested, pHy-CMVGFP and pHy-RPEGFP, both carrying the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) ubiquitous promoter or the RPE65-specific promoter, respectively. For the two amplicon vectors, the GFP reporter gene was efficiently expressed in retinal pigment epithelial (RPE) cells but not in the adjacent photoreceptors. GFP expression was maximum as early as 2 days post-administration but decreased over time to become almost undetectable at 6 weeks postinjection. Super-transduction with a second amplicon vector, pHSVlac, reactivated expression of GFP in approximately 10% of the cells initially transduced at 2 days postinjection of pHy-CMVGFP or pHy-RPEGFP. Reactivation of transgene expression was transient, no GFP signal was detected 8 days after pHSVlac injection. In conclusion, HSV-1 amplicon vectors allow rapid and efficient, but transient, gene transfer in RPE cells following subretinal injection.

Histological Analysis of GFP Expression in Murine Bone

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a beta-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.

Two Different Roles of Purified CD45 + c‐Kit + Sca‐1 + Lin − Cells After Transplantation in Muscles

Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)-labeled CD45+c-Kit+Sca-1+Lin- cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+c-Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+PAX7+, GFP+MyoD+, and GFP+Myf5+ satellite-like cells on the fibers. Single-fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.

Vertebrate Nonmuscle Myosin II Isoforms Rescue Small Interfering RNA-induced Defects in COS-7 Cell Cytokinesis

RNA interference (RNAi) treatment of monkey COS-7 cells, a cell line that lacks nonmuscle myosin heavy chain II-A (NMHC II-A) but contains NMHC II-B and II-C, was used to investigate the participation of NMHC isoforms in cytokinesis. We specifically suppressed the expression of NMHC II-B or II-C using 21 nucleotide small interfering RNA (siRNA) duplexes. Down-regulation of NMHC II-B protein expression to 10.2 +/- 0.7% inhibited COS-7 cell proliferation by 50% in the RNAi-treated cells compared with control cells. Moreover, whereas 8.7 +/- 1.0% of control cells were multinucleated, 62.4 +/- 8.8% of the NMHC II-B RNAi-treated cells were multinucleated 72 h after transfection. The RNAi-treated cells had increased surface areas and, unlike control cells, lacked actin stress fibers. Treatment of the COS-7 cells with NMHC II-C siRNA decreased NMHC II-C expression to 5.2 +/- 0.1% compared with the endogenous content of II-C; however, down-regulation of NMHC II-C did not cause increased multinucleation. Immunoblot analysis using a pan-myosin antibody showed that the content of NMHC II-C was less than one-twentieth the amount of NMHC II-B, thereby explaining the lack of response to II-C siRNA. Introducing green fluorescent protein (GFP)-tagged NMHC II isoforms into II-B siRNA-treated cells resulted in reduction of multinucleation from 62.4 +/- 8.8% to 17.8 +/- 2.2% using GFP-NMHC II-B, to 29.8 +/- 7.4% using GFP-NMHC II-A, and to 34.1 +/- 8.6% using NMHC II-C-GFP. These studies have shown that expression of endogenous NMHC II-C in COS-7 cells is insufficient for normal cytokinesis and that exogenous NMHC II-A and NMHC II-C can, at least partially, rescue the defect in cytokinesis due to the loss of NMHC II-B.

The Spatial Organization of Lipid Synthesis in the Yeast Saccharomyces cerevisiae Derived from Large Scale Green Fluorescent Protein Tagging and High Resolution Microscopy

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.

Isolation and molecular characterization of rbcS in the unicellular green alga Nannochloris bacillaris (Chlorophyta, Trebouxiophyceae)

SUMMARY The small subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carboxyase/oxygenase (Rubisco) is encoded by rbcS. We isolated and characterized three rbcS genes (NbrbcS1-1, NbrbcS1-2, and NbrbcS2) from the unicellular green alga Nannochloris bacillaris (Chlorophyta, Trebouxiophyceae). In the haploid N. bacillaris genome, each is a single-copy gene located on different chromosomes. Each mature peptide contains 140 amino acid residues. NbrbcS1-1 and NbrbcS1-2 are identical and share 80% identity with NbrbcS2, while the respective transit sequences share only approximately 48% identity with that of NbrbcS2. NbrbcS1-1 transcription was suppressed in the dark and recovered drastically after transfer to light. In contrast, NbrbcS1-2 and NbrbcS2 expression were not reduced after transfer from light to dark. In chlorophyllic tobacco cells containing green fluorescent protein fusion proteins of the transit sequences of each peptide, green fluorescent protein signals were localized on particles matching chloroplasts. The first introns of NbrbcS1-1 and NbrbcS1-2 are identical to the corresponding introns of 37 rbcS genes in eight embryophyte species. While the second intron is conserved in the green algae, the NbrbcS2 intron appears to involve sliding by one base pair. The NbrbcS1-1 and NbrbcS1-2 intron conserved in green algae and embryophytes might be an ancestral intron from nuclear-encoded rbcS.

Behavior of Transplanted Bone Marrow-derived GFP Mesenchymal Cells in Osteochondral Defect as a Simulation of Autologous Transplantation

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow-derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone.

Effect of herpesvirus infection on pancreatic duct cell secretion

To examine the effect of acute infection caused by herpesvirus (pseudorabies virus, PRV) on pancreatic ductal secretion. The virulent Ba-DupGreen (BDG) and non-virulent Ka-RREp0lacgfp (KEG) genetically modified strains of PRV were used in this study and both of them contain the gene for green fluorescent protein (GFP). Small intra/interlobular ducts were infected with BDG virus (10(7) PFU/mL for 6 h) or with KEG virus (10(10) PFU/mL for 6 h), while non-infected ducts were incubated only with the culture media. The ducts were then cultured for a further 18 h. The rate of HCO(3)(-) secretion (base efflux -J(B-)) was determined from the buffering capacity of the cells and the initial rate of intracellular acidification (1) after sudden blockage of basolateral base loaders with dihydro-4,4-diisothiocyanatostilbene-2,2-disulfonic acid (500 micromol/L) and amiloride (200 micromol/L), and (2) after alkali loading the ducts by exposure to NH(4)Cl. All the experiments were performed in HCO(3)(-)-buffered Ringer solution at 37 degrees (n = 5 ducts for each experimental condition). Viral structural proteins were visualized by immunohistochemistry. Virally-encoded GFP and immunofluorescence signals were recorded by a confocal laser scanning microscope. The BDG virus infected the majority of accessible cells of the duct as judged by the appearance of GFP and viral antigens in the ductal cells. KEG virus caused a similarly high efficiency of infection. After blockage of basolateral base loaders, BDG infection significantly elevated -J(B-) 24 h after the infection, compared to the non-infected group. However, KEG infection did not modify -J(B-). After alkali loading the ducts, -J(B-) was significantly elevated in the BDG group compared to the control group 24 h after the infection. As we found with the inhibitor stop method, no change was observed in the group KEG compared to the non-infected group. Incubation with the BDG or KEG strains of PRV results in an effective infection of ductal epithelial cells. The BDG strain of PRV, which is able to initiate a lytic viral cycle, stimulates HCO(3)(-) secretion in guinea pig pancreatic duct by about four- to fivefold, 24 h after the infection. However, the KEG strain of PRV, which can infect, but fails to replicate, has no effect on HCO(3)(-) secretion. We suggest that this response of pancreatic ducts to virulent PRV infection may represent a defense mechanism against invasive pathogens to avoid pancreatic injury.

A simple method for discriminating between cell membrane and cytosolic proteins

* Transgenic plants expressing either green fluorescent protein (GFP)-genomic DNA or GFP-cDNA fusions have been used as powerful tools to define the subcellular localization of many proteins. Because most plant cells are highly vacuolated, the cytosol is confined to a thin layer at the periphery of the cells, making it very difficult to distinguish among cell wall, cell membrane and cytosolic GFP-fusion proteins. * Plasmolysis tests inform about cell-wall localization of GFP-tagged proteins, but they do not discriminate between its cell membrane and/or cytoplasmic localization. By observing the GFP signal in transgenic protoplasts placed at a hypotonic solution, it was possible to distinguish between cell membrane and cytosolic GFP-tagged proteins. * The osmotic disruption of the protoplast vacuole in the hypotonic solution allows the diffusion of the GFP signal from the cell periphery to the central part of the cell volume when the GFP is fused to a soluble protein. By contrast, such diffusion does not occur when the protein under study is attached to the cell membrane. * The present method is easier, faster and cheaper than subcellular fractionating studies and/or immunoelectron microscopy, which have been traditionally used to discern between cell membrane and cytosolic proteins.

Hematopoietic Stem Cells Can Give Rise to Satellite-Like Cells in Skeletal Muscles.

Recent studies have indicated that bone marrow cells can regenerate damaged muscles, but also that they can adopt phenotype of other cells by cell fusion. It has also been reported that single hematopoietic stem cells (HSCs) can regenerate skeletal muscle although it is still controversial whether HSCs differentiate into satellite cells in muscle or not. In order to investigate the roles of HSCs in muscle regeneration and whether they can generate satellite cells or not, we purified and injected CD45+Lin−Sca-1+c-kit+(CD45+KSL) HSCs labeled by green fluorescent protein (GFP) into mice with or without irradiation. We examined time-course behavior of HSCs in recipient muscles with a fluorescent stereomicroscope and then immunohitochemical staining during the early and late phase after transplantation. Our direct visualization system gave evidence of massive GFP signals in all the muscles of only irradiated mice in early phase after transplantation. Transverse cryostat sections showed GFP+ Myosin+ muscle fibers along with numerous GFP+ hematopoietic cells in damaged muscle. We also found myogenin+GFP+ cells like myoblasts in very low number. These phenomena were temporal and GFP signals had dramatically reduced 30 days after transplantation. FISH analysis confirmed the GFP-DNAs in the nuclei of muscle fibers. These results suggested that most of GFP+HSCs fused with myofibers and participated in regeneration of damaged muscles, and a very few HSCs can differentiate into myoblast like cells expressing myogenin. After 6 months, GFP+ fibers could be hardly detected but GFP+c-Met+ mononuclear cells were located beneath the laminin+ basal lamina. Single fiber cultures from these mice showed proliferation of GFP+ fibers. These results suggested that HSC-derived cells settled beneath the basal lamina like satellite cells and might acquired the satellite cell activity.

Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention.

Mdc1/NFBD1 controls cellular responses to DNA damage, in part via interacting with the Mre11-Rad50-Nbs1 complex that is involved in the recognition, signalling, and repair of DNA double-strand breaks (DSBs). Here, we show that in live human cells, the transient interaction of Nbs1 with DSBs and its phosphorylation by ATM are Mdc1-independent. However, ablation of Mdc1 by siRNA or mutation of the Nbs1's FHA domain required for Mdc1 binding reduced the affinity of Nbs1 for DSB-flanking chromatin and caused aberrant pan-nuclear dispersal of Nbs1. This occurred despite normal phosphorylation of H2AX, indicating that lack of Mdc1 does not impair this DSB-induced chromatin change, but rather precludes the sustained engagement of Nbs1 with these regions. Mdc1 (but not Nbs1) became partially immobilized to chromatin after DSB generation, and siRNA-mediated depletion of H2AX prevented such relocalization of Mdc1 and uncoupled Nbs1 from DSB-flanking chromatin. Our data suggest that Mdc1 functions as an H2AX-dependent interaction platform enabling a switch from transient, Mdc1-independent recruitment of Nbs1 to DSBs towards sustained, Mdc1-dependent interactions with the surrounding chromosomal microenvironment.

In vivo detection and quantification of tetracycline by use of a whole-cell biosensor in the rat intestine.

An Escherichia coli biosensor strain, harboring the plasmid pTGFP2, was introduced into the gastrointestinal tract of gnotobiotic rats that continuously received drinking water containing tetracycline. Plasmid pTGFP2 contains a transcriptional fusion between a green fluorescent protein (GFP) gene and a tetracycline-regulated promoter and was shown to produce a proportional GFP signal in response to exposure to various tetracycline concentrations when harbored by an E. coli strain. The plasmid was highly unstable in the host bacteria colonizing the intestinal system of the animals, and rapid plasmid loss was observed. Reintroduction of the E. coli MC4100/pTGFP2 strain into animals already colonized by the plasmid-free E. coli strain the day before euthanasia made it possible to extract and analyze the biosensors from intestinal samples. The induction of GFP in the biosensor cells extracted from the animals was estimated on a single-cell basis by use of flow cytometry, and the mean induction of GFP in the samples was compared to a standard curve prepared from known tetracycline concentrations. The results showed that the bioavailable tetracycline concentration within the bacterial growth habitat of the intestine was proportional to the concentration of tetracycline in drinking water but represented only approximately 0.4% of the intake concentration. This is a significant finding which will help to clarify antimicrobial therapy in the intestinal environment.

Evaluation of the GFP signal and its aptitude for novel on-line monitoring strategies of recombinant fermentation processes

A high number of economically important recombinant proteins are produced in Escherichia coli based host/vector systems. The major obstacle for improving current processes is a lack of appropriate on-line in situ methods for the monitoring of metabolic burden and critical state variables. Here, a pre-evaluation of the reporter green fluorescent protein (GFP) was undertaken to assess its use as a reporter of stress associated promoter regulation. The investigation of GFP and its blue fluorescent variant BFP was done in model fermentations using E. coli HMS174(DE3)/pET11aGFPmut3.1and E. coli HMS174(DE3)/pET11aBFP host/vector systems cultured in fed-batch and chemostat regime. Our results prove the suitability of the fluorescent reporter proteins for the design of new strategies of on-line bioprocess monitoring. GFPmut3.1 variant can be detected after a short lag-phase of only 10 min, it shows a high fluorescence yield in relation to the amount of reporter protein, a good signal to noise ratio and a low detection limit. The fluorescence-signal and the amount of fluorescent protein, determined by ELISA, showed a close correlation in all fermentations performed. A combination of reporter technology with state of the art sensors helps to develop new strategies for efficient on-line monitoring needed for industrial process optimisation. The development of efficient monitoring will contribute to advanced control of recombinant protein production and accelerate the development of optimised production processes.

Differentiation trapping screen in live culture for genes expressed in cardiovascular lineages.

We have developed a gene trap vector that transduces an EGFP-neo fusion gene (Eno) to monitor the expression of trapped genes in living cells and embryos. Upon in vitro differentiation, most gene-trapped embryonic stem (ES) cell clones exhibited detectable green fluorescence in various specialized cell types, which can be followed in the live culture in real time. Populations of ES cell-derived cardiomyocytes, smooth muscle cells, vascular endothelial cells, and hematopoietic cells were readily recognized by their distinctive morphologies coupled with unique activities, allowing efficient screening for clones with trapped genes expressed in cardiovascular lineages. Applying G418 selection in parallel differentiation cultures further increased detection sensitivity and screening throughput by enriching reporter-expressing cells with intensified green fluorescent protein signals. Sequence analyses and chimera studies demonstrated that the expression of trapped genes in vivo closely correlated with the observed lineage specificity in vitro. This provides a strategy to identify and mutate genes expressed in lineages of interest for further functional studies.

Improved silencing vector co-expressing GFP and small hairpin RNA.

Small interfering RNA (siRNA) is a powerful tool for the specific silencing of gene expression. We developed an improved vector, pG-SUPER, that co-expresses green fluorescent protein (GFP) and small hairpin RNA simultaneously to facilitate analysis of silencing at the level of individual cells. As a test system, we analyzed lamin A/C knockdown in HeLa cells. The GFP signal was a reliable reporter (93%-98%) of strong knockdown (approximately 90%) over a wide range of GFP intensities. The GFP reporter made possible the application of fluorescent-activated cell sorting (FACS) to purify the knockdown cell population. Such populations facilitated Western blotting analysis to determine depletion of the target protein. pG-SUPER was also applied to evaluate gene replacement by exogenous genes rendered refractory to siRNA by introducing silent mutations. Recovery of lamin A was linearly correlated to the expression level of the rescue gene. pG-SUPER will expand plasmid-based siRNA applications through the easy and reliable detection of knockdown and rescued cells.

Bone marrow-derived cells do not incorporate into the adult growing vasculature.

Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into growing vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial growth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increase versus nonoccluded side, P<0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular growth by incorporating into vessel walls but may function as supporting cells.

Novel regulatory sequence -82/-62 functions as a key element to drive the somite-specificity of zebrafish myf-5.

Myf-5, a transcription factor that controls muscle differentiation, is expressed in somites during early embryogenesis. However, gene regulation of myf-5 is poorly understood and detailed functional analysis of the regulatory cis-elements is needed. In zebrafish, the myf-5 upstream sequence from -82 to -62 (-82/-62) was fused with a basal promoter and transferred to fertilized zebrafish eggs. The -82/-62 cassette drove green fluorescent protein (GFP) reporter gene expression specifically in the somites. Moreover, GFP signals were detected exclusively in the somites of 28-hpf embryos derived from eggs injected with pCMV-5x(-82/-62), which contained five copies of the -82/-62 cassette inserted within cytomegalovirus promoter/enhancer. Thus, the -82/-62 cassette, conserved in mouse myf-5, functions to drive somite-specific expression and to repress nonspecific expression during the early development of zebrafish embryos. Mutated sequence analysis of -82/-62 cassette showed that the -70/-62 sequence was the key element for controlling myf-5 specificity. The putative CCAAT-like box, located at -66/-62, could not direct somite-specific expression. A DNA-protein complex was specifically formed between the -70/-62 probe and embryonic nuclear extracts. We conclude that the -70/-62 motif is essential for controlling somite-specific expression and the CCAAT-like box is essential for activating gene transcription.