GFP Activity Research Articles

Matrix metalloproteinase 2 increases ROS in aortic tissue (1153.10)

So far ROS has been shown to activate MMP‐2, a key protease in cardiovascular diseases. This study tested whether MMP‐2´s effect on vascular dysfunction was mediated by ROS. The cDNA of the catalytic domain of the human MMP‐2 was cloned in fusion with GFP, and the activity of the hMMP‐2cat/GFP was assayed. The rhMMP‐2/GFP protein was catalytically active (both were expressed and purified from E coli). Both proteins were injected into the lumen of New Zealand rabbit aortas, and filled vessels (MMP‐2‐FA) were incubated for 30 min. Concentration used: 1.2 ug/ml or 16 mmol/L, 4 times the level of MMP‐2 found in healthy humans. The exogenous MMP‐2 increase in aortic wall was tracked by direct observation of GFP, immunostaining and gelatinolytic activity. This last parameter doubled in the MMP‐2‐FA, as well as in MMP‐2‐FA+PMSF (a serine‐proteinase inhibitor), but displayed normal values in MMP‐2‐FA+ Doxycycline (an MMP inhibitor). ROS activity was increased by 85% (±4%) in MMP‐2‐FA, while there was only a slight increase in the MMP‐2‐FA co‐infused with the MMP inhibitor Doxycycline (15 ±8%). Apocinin and PEG Catalase decreased the levels of ROS in MMP‐2‐FA to basal levels (P<0.001), while Tiron only reduced ROS levels by 17%(±8%)(p<0.05). This is the first study to show that MMP‐2 at levels described in plasma of patients are sufficient to increase ROS in vessels.Grant Funding Source: FAPESP

Neuroprotection by rAAV-mediated gene transfer of bone morphogenic protein 7

BackgroundBone morphogenic proteins (BMPs) promote the survival of neurons, suggesting a therapeutic application of BMPs in the treatment of acute and chronic neurodegenerative disorders. However, the application of recombinant BMPs in vivo is limited by their short half-life. To provide a continuous supply for functionally active BMPs, we expressed BMP7, BMP2 and the BMP inhibitor Noggin under the control of rAAV vectors in vivo. For visual control of rAAV-mediated BMP (v-BMP) expression we fused the secreted morphogenic polypeptides and the fluorescent reporter protein Venus via the ‘ribosomal skip’ promoting 2A peptide-bridge.ResultsIn primary cortical neurons, the rAAV-expressed morphogenic polypeptides were efficiently released from the 2A-Venus fusion precursors, were secreted, correctly processed and functionally active as shown by their effects on Smad phosphorylation in HeLa cells and in primary neurons, by the protection of v-BMP7-transduced primary cortical neurons against oxidative stress, and by the activation of BMP responsive GFP in v-BMP2 transduced reporter mice. In the stroke model of middle cerebral artery occlusion rAAV-transduced v-BMP7 reduced the infarct size in mice.ConclusionPolycistronic rAAV vectors encoding secreted polypeptides and 2A-linked reporter proteins are potential novel therapeutic tools for the treatment of neurological and neurodegenerative diseases. Using this technique we documented that rAAV delivery of BMP7 reduced ischemic cell death in mice.

Genetic transformation of Eucalyptus globulus using the vascular-specific EgCCR as an alternative to the constitutive CaMV35S promoter

Strong constitutive promoters, such as CaMV35S, are widely used for plant transformation, but undesirable phenotypic changes have been reported when used to drive biotic stress tolerance and/or for modifying lignin content. The promoter of the eucalyptus cinnamoyl CoA reductase (CCR), a key enzyme of the lignin biosynthetic pathway, was shown to be preferentially expressed in vascular tissues both in herbaceous and woody transgenic plants but not eucalyptus. In this work, we transformed Eucalyptus globulus with the EgCCR promoter governing both β-glucuronidase (GUS) and GFP activity patterns. No statistical differences were found between the survival rate and percentage of GUS positive shoots between eucalyptus transformed with either the constitutive CaMV35Sor with the EgCCR promoter. The EgCCR transformed plantlets exhibited high GUS expression levels associated with the vascular tissues opening the possibility of targeting vascular-associated traits such as lignin content or vascular pathogen resistance in adult elite plants of eucalyptus while avoiding the undesirable pleiotropic effects caused by strong constitutive promoters.

Genetic Transformation of the Obligate ParasitePlasmodiophora brassicae

A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem.

Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus. The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public's acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6% transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100% deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8% of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.

Two Types of Tet-On Transgenic Lines for Doxycycline-Inducible Gene Expression in Zebrafish Rod Photoreceptors and a Gateway-Based Tet-On Toolkit

The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA), one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE)-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research.

Remote activation of biomolecules in deep tissues using near-infrared-to-UV upconversion nanotransducers

Controlled activation or release of biomolecules is very crucial in various biological applications. Controlling the activity of biomolecules have been attempted by various means and controlling the activity by light has gained popularity in the past decade. The major hurdle in this process is that photoactivable compounds mostly respond to UV radiation and not to visible or near-infrared (NIR) light. The use of UV irradiation is limited by its toxicity and very low tissue penetration power. In this study, we report the exploitation of the potential of NIR-to-UV upconversion nanoparticles (UCNs), which act as nanotransducers to absorb NIR light having high tissue penetration power and negligible phototoxicity and emit UV light locally, for photoactivation of caged compounds and, in particular, used for photo-controlled gene expression. Both activation and knockdown of GFP was performed in both solution and cells, and patterned activation of GFP was achieved successfully by using upconverted UV light produced by NIR-to-UV UCNs. In-depth photoactivation through tissue phantoms and in vivo activation of caged nucleic acids were also accomplished. The success of this methodology has defined a unique level in the field of photo-controlled activation and delivery of molecules.

Exploring the Function of Alcohol Dehydrogenases During the Endophytic Life of Azoarcus Sp. Strain BH72

The endophytic bacterium Azoarcus sp. strain BH72 is capable of colonizing the interior of rice roots, where it finds suitable physicochemical properties for multiplying and fixing nitrogen. Because these properties are poorly understood, a microtiter-plate-based screening of a transcriptional gfp (green fluorescent protein) fusion library of Azoarcus sp. grown under different conditions was performed. Monitoring of the GFP activity allowed the identification of a gene highly expressed in medium supplemented with ethanol. Sequence analysis revealed that this gene encodes a pyrrolo-quinoline quinone-dependent alcohol dehydrogenase (ADH). Inspection of the complete genome sequence of the Azoarcus sp. strain BH72 identified seven additional genes encoding putative ADH, indicating that BH72 is well equipped to survive in different environmental conditions offering various alcohols as carbon source. Analyses of these eight putative ADH showed that expression of three was induced by ethanol, of which two were also expressed inside rice roots. The fact that waterlogged plants such as rice accumulate ethanol suggests that ethanol occurs in sufficiently high concentration within the root to induce expression of bacterial ADH. Disruption of these two ADH evoked a reduced competitiveness to the wild type in colonizing rice roots internally. Thus, it is likely that ethanol is an important carbon source for the endophytic life of Azoarcus sp.

Transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) by agitation with glass beads

A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P. subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable conditions for production of viable protoplasts were pH 6.5, 25 degrees C, and 3 h of enzyme treatment. The protoplast yield was 61.2% when P. subcordiformis cells were added to the enzyme solution at a concentration of 10(7) cell ml(-1). The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency was about 10(-5), and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis.

Determining GFP Activity in HCT-8 Cells Infected with Cryptosporidium Parvum by Reverse Transcription Polymerase Chain Reaction and Nested PCR

ABSTRACTThis study focusses on the determination of GFP activity in HCT-8 cells infected with Cryptosporidium parvum measured by RT-PCR and Nested PCR. Our findings demonstrate that the level of expression for green fluorescent protein reporter vector was undetecteable under fluorescent microscope at 48 h postelectroporation in the presence of 25 μg closed-circular construct. Real-time melting curves and quantitation analysis of RT PCR and Nested PCR were used to show low mRNA expression of GFP. The methods used here allow detection of GFP activity in HCT-8 cells.Here we describe the preliminary study which forms the basis for future transient transformation of C. parvum. These approaches may contribute to our understanding of the feasibility of transforming C. parvum, since the ability to transiently transform C. parvum should lead to expanded research on this important parasite.

Expression of a gfp gene in Penicillium nordicum under control of the promoter of the ochratoxin A polyketide synthase gene

A gfp reporter gene strain of Penicillium nordicum was constructed in order to be able to study the influence of environmental parameters on ochratoxin A biosynthesis. To introduce the gfp gene an Agrobacterium tumefaciens mediated transformation system (ATMT) for P. nordicum was established resulting in a transformation efficiency of about 60 transformants per µg of DNA. The selection principle was based on the hygromycin B resistance gene located on the TI-DNA fragment of the binary vector system. PCR and Southern blot hybridization revealed that the TI-DNA was integrated into the chromosome of P. nordicum. To show that the GFP protein can be used as a reporter gene in P. nordicum, this species was subsequently transformed with a vector, carrying a gfp gene under the control of the strong constitutive gpd promoter of A. nidulans. Moreover in this vector construction the gfp gene contained a stuA nuclear localization domain. Successful transformed strains showed a strong GFP activity located in the nuclei after light stimulation in contrast to the wild type which showed only very weak unspecific auto fluorescence under these conditions. Based on this proof of principle a vector was constructed in which the promoter of the otapksPN gene, the gene of the ochratoxin A polyketide synthase of P. nordicum was placed in front of the gfp gene. This construct was transformed into P. nordicum by ATMT and the resulting transformants were analysed by fluorescence microscopy. In these transformants the whole mycelial cells showed GFP activity after light stimulation, whereas the wild type strain did not. When the transformed strains were grown on medium which suppressed ochratoxin A biosynthesis, a very low level of fluorescence could be detected, whereas a high level of fluorescence was seen after growth on medium supportive for ochratoxin A biosynthesis.

Probing Nucleocytoplasmic Transport with Fluorescence Fluctuation Spectroscopy and Two-photon Activation of Photoactivable GFP

Large proteins and macromolecular complexes have to enter and leave the nucleus in an efficient and selective manner. Macromolecules that are greater than 40 kD are transported actively across the nuclear envelope through nuclear pore complexes using soluble transport factors or carrier molecules that cycle between the cytoplasm and nucleus. The carrier proteins themselves interact with each other in order to transport cargo proteins across the nuclear pore complexes. In this work, we apply dual-color time-integrated fluorescence cumulant analysis (TIFCA), a fluorescence fluctuation spectroscopy technique, to investigate the protein interactions of the carrier proteins directly in cells. In addition, we apply two-photon activation to directly examine the nucleocytoplasmic transport of photoactivable GFP tagged carrier proteins. With these two approaches, we are able to probe the nucleocytoplasmic transport process of NTF2 directly inside cells and under equilibrium conditions. We investigate the oligomerization of NTF2 in cells and its transport properties when crossing the nuclear pore complexes.The experiments identify the presence of NTF2 dimers in the cytoplasm and nucleus of cells, while the measured transport properties across the nuclear pore complex are not consistent with dimeric NTF2. We will discuss the implications of these results for functions of nuclear envelope and models of nucleocytoplasmic transport. This work is supported by the National Science Foundation (PHY-0346782) and NIH grant R01GM064589.

Cre reporter mouse expressing a nuclear localized fusion of GFP and β‐galactosidase reveals new derivatives of Pax3‐expressing precursors

A new Cre-reporter strain of mouse has been developed that expresses a fusion protein derived from the lacZ gene fused to GFP with a nuclear localization signal. This construct is expressed from the ROSA26 locus upon Cre-mediated recombination that removes a loxP-flanked PGK-neo cassette, thus allowing for detection of Cre activity in all tissues. This reporter strain, which is similar to prior R26R and R26EGFP strains, has certain advantages related to the nuclear expression and the combined expression of both beta-galactosidase and GFP activities. We show that the use of this newly developed reporter line allows for enhanced resolution, detection and co-localization. Thus, we report a previously unrecognized subset of venous endothelial cells derived from Pax3 expressing precursors.

Keap1/Nrf2 Signaling Regulates Oxidative Stress Tolerance and Lifespan in Drosophila

Keap1/Nrf2 signaling defends organisms against the detrimental effects of oxidative stress and has been suggested to abate its consequences, including aging-associated diseases like neurodegeneration, chronic inflammation, and cancer. Nrf2 is a prominent target for drug discovery, and Nrf2-activating agents are in clinical trials for cancer chemoprevention. However, aberrant activation of Nrf2 by keap1 somatic mutations may contribute to carcinogenesis and promote resistance to chemotherapy. To evaluate potential functions of Keap1 and Nrf2 for organismal homeostasis, we characterized the pathway in Drosophila. We demonstrate that Keap1/Nrf2 signaling in the fruit fly is activated by oxidants, induces antioxidant and detoxification responses, and confers increased tolerance to oxidative stress. Importantly, keap1 loss-of-function mutations extend the lifespan of Drosophila males, supporting a role for Nrf2 signaling in the regulation of longevity. Interestingly, cancer chemopreventive drugs potently stimulate Drosophila Nrf2 activity, suggesting the fruit fly as an experimental system to identify and characterize such agents.

Oryza Tag Line , a phenotypic mutant database for the Génoplante rice insertion line library

To organize data resulting from the phenotypic characterization of a library of 30 000 T-DNA enhancer trap (ET) insertion lines of rice (Oryza sativa L cv. Nipponbare), we developed the Oryza Tag Line (OTL) database (http://urgi.versailles.inra.fr/OryzaTagLine/). OTL structure facilitates forward genetic search for specific phenotypes, putatively resulting from gene disruption, and/or for GUSA or GFP reporter gene expression patterns, reflecting ET-mediated endogenous gene detection. In the latest version, OTL gathers the detailed morpho-physiological alterations observed during field evaluation and specific screens in a first set of 13 928 lines. Detection of GUS or GFP activity in specific organ/tissues in a subset of the library is also provided. Search in OTL can be achieved through trait ontology category, organ and/or developmental stage, keywords, expression of reporter gene in specific organ/tissue as well as line identification number. OTL now contains the description of 9721 mutant phenotypic traits observed in 2636 lines and 1234 GUS or GFP expression patterns. Each insertion line is documented through a generic passport data including production records, seed stocks and FST information. 8004 and 6101 of the 13 928 lines are characterized by at least one T-DNA and one Tos17 FST, respectively that OTL links to the rice genome browser OryGenesDB.

Prednisolone mediated suppression of HIV-1 viral load strongly correlates with C-C chemokine CCL2: In vivo and in vitro findings

CCL2 (MCP-1) is a proinflammatory chemokine induced in HIV-1 infection. We have previously demonstrated a significant correlation of CCL2 gene expression with HIV-1 viremia. In this study we investigated the effect of prednisolone on CCL2 gene expression and viral load in an HIV-1-infected patient receiving high-dose prednisolone for severe uveitis. We observed a > 1 log reduction of HIV-1 viral load, associated with more than hundred fold reduction of CCL2 expression at day 3 of prednisolone treatment. In vitro HIV-1 infection of PBMC demonstrated reduced HIV-1 replication in the presence of prednisolone. Flow cytometric analysis revealed 50% reduction of LTR driven GFP activity by prednisolone in GHOST cells. These findings indicate that prednisolone suppresses both HIV-1 viral load and CCL2 mRNA expression, an association which might be exploited for future anti-inflammatory therapeutic strategies in HIV-1 infection.

In Vivo and in Vitro Investigation of Transcriptional Regulation by DntR

DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.

High expression of transgene protein in Spirodela

The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA-DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant.

Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering

BackgroundNowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthetic promoters of Escherichia coli as a useful tool for fine tuning gene expression is discussed here.ResultsA degenerated oligonucleotide sequence that encodes consensus sequences for E. coli promoters separated by spacers of random sequences has been designed and synthesized. This 57 bp long sequence contains 24 conserved, 13 semi-conserved (W, R and D) and 20 random nucleotides. This mixture of DNA fragments was cloned into a promoter probing vector (pVIK165). The ligation mixtures were transformed into competent E. coli MA8 and the resulting clones were screened for GFP activity by measuring the relative fluorescence units; some clones produced high fluorescence intensity, others weak fluorescence intensity. The clones cover a range of promoter activities from 21.79 RFU/OD600 ml to 7606.83 RFU/OD600 ml. 57 promoters were sequenced and used for promoter analysis. The present results conclusively show that the postulates, which link promoter strength to anomalies in the -10 box and/or -35 box, and to the length of the spacer, are not generally valid. However, by applying Partial Least Squares regression, a model describing the promoter strength was built and validated.ConclusionFor Escherichia coli, the promoter strength can not been linked to anomalies in the -10 box and/or -35 box, and to the length of the spacer. Also a probabilistic approach to relate the promoter sequence to its strength has some drawbacks. However, by applying Partial Least Squares regression, a good correlation was found between promoter sequence and promoter strength. This PLS model can be a useful tool to rationally design a suitable promoter in order to fine tune gene expression.

Macrophage colony stimulating factor is a crucial factor for the intrinsic macrophage response in mice heterozygously deficient for the myelin protein P0

Mouse mutants heterozygously deficient for the myelin protein P0 (P0+/−) resemble certain forms of human hereditary neuropathies. Endoneurial macrophages of intrinsic origin are intimately involved in the pathogenesis of the demyelinating neuropathy in these mutants. We have previously shown that deficiency for macrophage colony stimulating factor (M-CSF) prevents an increase of the number of endoneurial macrophages and alleviates the mutants' demyelinating phenotype. The aim of this study was to investigate which population of endoneurial macrophages – long-term resident macrophages or recently infiltrated macrophages – is affected by M-CSF deficiency. For this purpose, we generated bone marrow chimeric mice by transplanting GFP+ bone marrow into P0 mutants (P0+/−) and P0 mutants that lack M-CSF (P0+/− mcsf-op). This enabled us to discriminate recently infiltrated short-term resident GFP+ macrophages from long-term resident GFP− macrophages. Three months after bone marrow transplantation, P0+/− mice expressing M-CSF showed a substantial upregulation and activation of both GFP− and GFP+ macrophages in femoral nerves when compared to P0+/+ mice. In contrast, in P0+/− mcsf-op mutants, both GFP− and GFP+ macrophages did not substantially increase. Only small numbers of GFP+ but no GFP− macrophages were activated and phagocytosed myelin in chimeric P0+/− mcsf-op mutants, possibly reflecting recent activation outside the endoneurium before entering the nerve. Our findings demonstrate that M-CSF is crucial for the activation, in situ increase and myelin phagocytosis of both long-term and short-term resident endoneurial macrophages in P0+/− myelin mutants. M-CSF is, therefore, considered as a target candidate for therapeutic strategies to treat human demyelinating neuropathies.