pdsred express n1 plasmid  (TaKaRa)

 
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    Name:
    pDsRed Express N1 Vector
    Description:
    DsRed Express and DsRed Express2 are so called because they are rapidly maturing variants of Discosoma sp red fluorescent protein DsRed They also demonstrate enhanced solubility DsRed Express2 with the higher solubility reduced green emission and faster maturation rate than the original DsRed or DsRed2
    Catalog Number:
    632429
    Price:
    None
    Size:
    20 ug
    Category:
    DsRed Express and DsRed Express2 fluorescent proteins Red fluorescent proteins Fluorescent protein plasmids Fluorescent proteins Gene function
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    Structured Review

    TaKaRa pdsred express n1 plasmid
    DsRed Express and DsRed Express2 are so called because they are rapidly maturing variants of Discosoma sp red fluorescent protein DsRed They also demonstrate enhanced solubility DsRed Express2 with the higher solubility reduced green emission and faster maturation rate than the original DsRed or DsRed2
    https://www.bioz.com/result/pdsred express n1 plasmid/product/TaKaRa
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pdsred express n1 plasmid - by Bioz Stars, 2020-07
    93/100 stars

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    Related Articles

    Clone Assay:

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: .. RFP-nls was cloned into pCMV5-T7b (derivative of pCMV5 with T7 tag, D. Russell, University of Texas Southwestern) by using NotI and EcoRI to introduce the 3’ nuclear localization signals of RPS6/S6 ribosomal subunit, and EcoRI and NheI to place the RFP (pDS-Red N1; Clontech Laboratories, 632429) in frame 5’ of the nuclear localization signal, similarly previously GFP-nls was generated. .. Cloning of mouse Tomm22 ( ) was performed by extracting RNA from wild-type hindlimb muscle using TRIzol Reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer's instructions, reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs, M0253), amplified by PCR (for primers see below), and cloned in EcoRV-digested pBluescript SK+ (Agilent Technologies, 212205).

    Article Title: Rab1a rescues the toxicity of PRAF3
    Article Snippet: .. For the hPRAF3-DsRed construct, PCR fragment for hPRAF3 was directly cloned into pDsRed-Express-N1 vector (Clontech Laboratories, CA) as previously mentioned . hPRAF3-GFP expression construct was manufactured from the sub-cloned vector in pDONR221 as described above through LR recombination with pcDNA-DEST47 (Thermo Fisher Scientific, MA). .. 2.2 Yeast growth test The combinations of expression vectors used for the growth test are summarised in .

    Transfection:

    Article Title: Mouse Embryonic Stem Cells, but Not Somatic Cells, Predominantly Use Homologous Recombination to Repair Double-Strand DNA Breaks
    Article Snippet: .. Briefly, 5 × 106 cells were transfected in PBS with 40 μg of construct DNA and cotransfected with 5 μg pDsRed Express N1 (Clontech) using settings of 400 V and 250 μF. ..

    Article Title: Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy
    Article Snippet: .. Seven micrograms/well of pDsRed-Express-N1 (Clontech) was simultaneously transfected as verification of siRNA delivery. .. Seventy two hours later, the cells were treated with Premo™ Autophagy Sensors (LC3B-GFP) for 16 hours and analyzed for the presence of LC3 puncta using confocal microscopy.

    Article Title: Heterologous expression of porcine elongase 6 (ELOVL6) gene in a human cell line
    Article Snippet: .. In vector control, (Fig. – ) cells transfected with pDsRed-Express-N1 displayed both red (Fig. and ) and blue (Fig. and ) fluorescence for functional DsRed protein along with the characteristic DAPI in nuclei. .. In the treatment group, (Fig. – ) cells transfected with PCMV -Elongase6-DsRed exhibited characteristic blue fluorescence (Fig. and ) for DAPI, red fluorescence (Fig. and ) for DsRed and green fluorescence (Fig. and ) for porcine Elongase6 protein.

    Expressing:

    Article Title: Rab1a rescues the toxicity of PRAF3
    Article Snippet: .. For the hPRAF3-DsRed construct, PCR fragment for hPRAF3 was directly cloned into pDsRed-Express-N1 vector (Clontech Laboratories, CA) as previously mentioned . hPRAF3-GFP expression construct was manufactured from the sub-cloned vector in pDONR221 as described above through LR recombination with pcDNA-DEST47 (Thermo Fisher Scientific, MA). .. 2.2 Yeast growth test The combinations of expression vectors used for the growth test are summarised in .

    Fluorescence:

    Article Title: Heterologous expression of porcine elongase 6 (ELOVL6) gene in a human cell line
    Article Snippet: .. In vector control, (Fig. – ) cells transfected with pDsRed-Express-N1 displayed both red (Fig. and ) and blue (Fig. and ) fluorescence for functional DsRed protein along with the characteristic DAPI in nuclei. .. In the treatment group, (Fig. – ) cells transfected with PCMV -Elongase6-DsRed exhibited characteristic blue fluorescence (Fig. and ) for DAPI, red fluorescence (Fig. and ) for DsRed and green fluorescence (Fig. and ) for porcine Elongase6 protein.

    Mutagenesis:

    Article Title: Conformational activation of talin by RIAM triggers integrin-mediated cell adhesion
    Article Snippet: .. Full length RIAM fused to EGFP was a kind gift from Dr Frank Gertler at MIT and was used to subclone RIAM 1-306 and the RIAM mutant into EGFP or dsRED vector, pDsRed-Express-N1 (Clontech Lab, Mountain View, CA). ..

    Construct:

    Article Title: Mouse Embryonic Stem Cells, but Not Somatic Cells, Predominantly Use Homologous Recombination to Repair Double-Strand DNA Breaks
    Article Snippet: .. Briefly, 5 × 106 cells were transfected in PBS with 40 μg of construct DNA and cotransfected with 5 μg pDsRed Express N1 (Clontech) using settings of 400 V and 250 μF. ..

    Article Title: Rab1a rescues the toxicity of PRAF3
    Article Snippet: .. For the hPRAF3-DsRed construct, PCR fragment for hPRAF3 was directly cloned into pDsRed-Express-N1 vector (Clontech Laboratories, CA) as previously mentioned . hPRAF3-GFP expression construct was manufactured from the sub-cloned vector in pDONR221 as described above through LR recombination with pcDNA-DEST47 (Thermo Fisher Scientific, MA). .. 2.2 Yeast growth test The combinations of expression vectors used for the growth test are summarised in .

    Functional Assay:

    Article Title: Heterologous expression of porcine elongase 6 (ELOVL6) gene in a human cell line
    Article Snippet: .. In vector control, (Fig. – ) cells transfected with pDsRed-Express-N1 displayed both red (Fig. and ) and blue (Fig. and ) fluorescence for functional DsRed protein along with the characteristic DAPI in nuclei. .. In the treatment group, (Fig. – ) cells transfected with PCMV -Elongase6-DsRed exhibited characteristic blue fluorescence (Fig. and ) for DAPI, red fluorescence (Fig. and ) for DsRed and green fluorescence (Fig. and ) for porcine Elongase6 protein.

    Introduce:

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: .. RFP-nls was cloned into pCMV5-T7b (derivative of pCMV5 with T7 tag, D. Russell, University of Texas Southwestern) by using NotI and EcoRI to introduce the 3’ nuclear localization signals of RPS6/S6 ribosomal subunit, and EcoRI and NheI to place the RFP (pDS-Red N1; Clontech Laboratories, 632429) in frame 5’ of the nuclear localization signal, similarly previously GFP-nls was generated. .. Cloning of mouse Tomm22 ( ) was performed by extracting RNA from wild-type hindlimb muscle using TRIzol Reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer's instructions, reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs, M0253), amplified by PCR (for primers see below), and cloned in EcoRV-digested pBluescript SK+ (Agilent Technologies, 212205).

    Generated:

    Article Title: In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy
    Article Snippet: .. RFP-nls was cloned into pCMV5-T7b (derivative of pCMV5 with T7 tag, D. Russell, University of Texas Southwestern) by using NotI and EcoRI to introduce the 3’ nuclear localization signals of RPS6/S6 ribosomal subunit, and EcoRI and NheI to place the RFP (pDS-Red N1; Clontech Laboratories, 632429) in frame 5’ of the nuclear localization signal, similarly previously GFP-nls was generated. .. Cloning of mouse Tomm22 ( ) was performed by extracting RNA from wild-type hindlimb muscle using TRIzol Reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer's instructions, reverse transcribed with M-MuLV Reverse Transcriptase (New England Biolabs, M0253), amplified by PCR (for primers see below), and cloned in EcoRV-digested pBluescript SK+ (Agilent Technologies, 212205).

    Cell Culture:

    Article Title: T-type Ca2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling
    Article Snippet: .. GCaMP imaging in superior cervical ganglion neurons SCG neurons were co-transfected with either 0.8 µg pCAGGs-mCherry (Addgene) or 0.8 µg pDsRed-Express-N1 (Clontech) and 2.5 µg membrane-directed pGPCMV-GCaMP6s-CAAX (Addgene) using a microporator (Digital Bio; 2 × 30 ms pulse at 900 V) and cultured at a high density in medium containing 10 ng ml−1 NGF and 300 nM TAPI-O on glass coverslips that had been treated with polyornithine and laminin. .. After 24 h, the coverslips were mounted in a laminar-flow perfusion and stimulation chamber (Warner Instruments) on the stage of an epifluorescence microscope (Axiovert 200M, Zeiss).

    Mass Spectrometry:

    Article Title: T-type Ca2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling
    Article Snippet: .. GCaMP imaging in superior cervical ganglion neurons SCG neurons were co-transfected with either 0.8 µg pCAGGs-mCherry (Addgene) or 0.8 µg pDsRed-Express-N1 (Clontech) and 2.5 µg membrane-directed pGPCMV-GCaMP6s-CAAX (Addgene) using a microporator (Digital Bio; 2 × 30 ms pulse at 900 V) and cultured at a high density in medium containing 10 ng ml−1 NGF and 300 nM TAPI-O on glass coverslips that had been treated with polyornithine and laminin. .. After 24 h, the coverslips were mounted in a laminar-flow perfusion and stimulation chamber (Warner Instruments) on the stage of an epifluorescence microscope (Axiovert 200M, Zeiss).

    Polymerase Chain Reaction:

    Article Title: Rab1a rescues the toxicity of PRAF3
    Article Snippet: .. For the hPRAF3-DsRed construct, PCR fragment for hPRAF3 was directly cloned into pDsRed-Express-N1 vector (Clontech Laboratories, CA) as previously mentioned . hPRAF3-GFP expression construct was manufactured from the sub-cloned vector in pDONR221 as described above through LR recombination with pcDNA-DEST47 (Thermo Fisher Scientific, MA). .. 2.2 Yeast growth test The combinations of expression vectors used for the growth test are summarised in .

    Plasmid Preparation:

    Article Title: Rab1a rescues the toxicity of PRAF3
    Article Snippet: .. For the hPRAF3-DsRed construct, PCR fragment for hPRAF3 was directly cloned into pDsRed-Express-N1 vector (Clontech Laboratories, CA) as previously mentioned . hPRAF3-GFP expression construct was manufactured from the sub-cloned vector in pDONR221 as described above through LR recombination with pcDNA-DEST47 (Thermo Fisher Scientific, MA). .. 2.2 Yeast growth test The combinations of expression vectors used for the growth test are summarised in .

    Article Title: Heterologous expression of porcine elongase 6 (ELOVL6) gene in a human cell line
    Article Snippet: .. In vector control, (Fig. – ) cells transfected with pDsRed-Express-N1 displayed both red (Fig. and ) and blue (Fig. and ) fluorescence for functional DsRed protein along with the characteristic DAPI in nuclei. .. In the treatment group, (Fig. – ) cells transfected with PCMV -Elongase6-DsRed exhibited characteristic blue fluorescence (Fig. and ) for DAPI, red fluorescence (Fig. and ) for DsRed and green fluorescence (Fig. and ) for porcine Elongase6 protein.

    Article Title: Conformational activation of talin by RIAM triggers integrin-mediated cell adhesion
    Article Snippet: .. Full length RIAM fused to EGFP was a kind gift from Dr Frank Gertler at MIT and was used to subclone RIAM 1-306 and the RIAM mutant into EGFP or dsRED vector, pDsRed-Express-N1 (Clontech Lab, Mountain View, CA). ..

    Imaging:

    Article Title: T-type Ca2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling
    Article Snippet: .. GCaMP imaging in superior cervical ganglion neurons SCG neurons were co-transfected with either 0.8 µg pCAGGs-mCherry (Addgene) or 0.8 µg pDsRed-Express-N1 (Clontech) and 2.5 µg membrane-directed pGPCMV-GCaMP6s-CAAX (Addgene) using a microporator (Digital Bio; 2 × 30 ms pulse at 900 V) and cultured at a high density in medium containing 10 ng ml−1 NGF and 300 nM TAPI-O on glass coverslips that had been treated with polyornithine and laminin. .. After 24 h, the coverslips were mounted in a laminar-flow perfusion and stimulation chamber (Warner Instruments) on the stage of an epifluorescence microscope (Axiovert 200M, Zeiss).

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  • 88
    TaKaRa monomeric dsred
    Cx32/Cx30 and Cx47/Cx43 form functional channels. Neuro2A cells were transiently transfected with <t>pIRES2-EGFP</t> or <t>pIRES2-DsRed</t> vectors containing Cx30, Cx32, Cx43, Cx47, or no insert [vector alone control (CTR)]. After 24 h, EGFP- and DsRed-expressing cells were mixed in a 1 to 1 ratio; pairs were assessed by dual whole-cell patch clamping 6–48 h later. For each combination listed on the x -axis, the mean and SE of the G j and number of pairs tested is shown. Using a Kruskal–Wallis test followed by Dunn's multiple comparison test, only Cx32/Cx30 and Cx47/Cx43 channels have G j that is significantly greater (* p
    Monomeric Dsred, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monomeric dsred/product/TaKaRa
    Average 88 stars, based on 4 article reviews
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    85
    TaKaRa dna damage conditions pdsred monomer n1 pdsred plasmid
    Gene inactivation by oxidative guanine lesions. ( A ) Dot density plots obtained by flow-cytometric analyses of the expression of DsRed-Monomer and green fluorescent protein (GFP) in HeLa cells at indicated times after co-transfection with equal amounts of pZA and <t>pDsRed</t> plasmids. The plasmid encoding for GFP was either not damaged (oG = 0) or contained on average three oxidized guanines (oG = 3). The transfected cells that do not express GFP localize in the upper left quadrants (marked with asterisks). ( B ) Quantitative analyses of the data presented in A representing percentages of the transfected cells located in the upper left quadrants (left) and median GFP fluorescence in the cells with a high DsRed-Monomer expression level (right). ( C ) Detection of oxidized guanine bases (oG) in the damaged plasmids by incubation with Fpg <t>DNA</t> glycosylase. The numbers of oxidized guanines were determined from the ratios of covalently closed circular (ccc) to open circular (oc) forms as described in ‘Materials and Methods’ section.
    Dna Damage Conditions Pdsred Monomer N1 Pdsred Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna damage conditions pdsred monomer n1 pdsred plasmid/product/TaKaRa
    Average 85 stars, based on 1 article reviews
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    90
    TaKaRa gl261 murine glioma cells expressing dsred2
    QD655 can be used in combination with fluorescent proteins. (A-B). <t>DsRed2</t> (green) and QD655 (red) emission spectra under an 800nm NLO (A) or 1050nm OPO (B) excitation. (C). Spectrally unmixed image of <t>GL261</t> DsRed2-expressing cells (green) growing on a
    Gl261 Murine Glioma Cells Expressing Dsred2, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gl261 murine glioma cells expressing dsred2/product/TaKaRa
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gl261 murine glioma cells expressing dsred2 - by Bioz Stars, 2020-07
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    92
    TaKaRa plasmid encoding dsred
    GH attenuates endogenous NHEJ DNA repair by inhibiting DNA-PKcs phosphorylation. ( A ) hNCC containing a chromosomally integrated NHEJ reporter cassette were cotransfected with I-SceI and <t>DsRed</t> expression vectors and treated with 500 ng/ml GH overnight. The intact reporters are negative for GFP. Upon induction of a DSB by I-SceI digestion, the functional GFP gene is reconstituted. Cells were also transfected with <t>pDsRed2-N1</t> as transfection control, and the percent of DsRed + cells indicates transfection efficiency. Cells were analyzed by FACS on day 5 after GH treatment, and the relative efficiency of DNA DSB repair was calculated as the ratio of GFP + cells/DsRed + cells. Representative analysis is shown. ( B ) Graph shows fold change in GFP positivity ± SEM in 5 independent assays. Controls represent cells not treated with GH. Differences were assessed with Tukey-adjusted mixed model regression. ( C .
    Plasmid Encoding Dsred, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 2 article reviews
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    plasmid encoding dsred - by Bioz Stars, 2020-07
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    Cx32/Cx30 and Cx47/Cx43 form functional channels. Neuro2A cells were transiently transfected with pIRES2-EGFP or pIRES2-DsRed vectors containing Cx30, Cx32, Cx43, Cx47, or no insert [vector alone control (CTR)]. After 24 h, EGFP- and DsRed-expressing cells were mixed in a 1 to 1 ratio; pairs were assessed by dual whole-cell patch clamping 6–48 h later. For each combination listed on the x -axis, the mean and SE of the G j and number of pairs tested is shown. Using a Kruskal–Wallis test followed by Dunn's multiple comparison test, only Cx32/Cx30 and Cx47/Cx43 channels have G j that is significantly greater (* p

    Journal: The Journal of Neuroscience

    Article Title: Two Distinct Heterotypic Channels Mediate Gap Junction Coupling between Astrocyte and Oligodendrocyte Connexins

    doi: 10.1523/JNEUROSCI.3395-07.2007

    Figure Lengend Snippet: Cx32/Cx30 and Cx47/Cx43 form functional channels. Neuro2A cells were transiently transfected with pIRES2-EGFP or pIRES2-DsRed vectors containing Cx30, Cx32, Cx43, Cx47, or no insert [vector alone control (CTR)]. After 24 h, EGFP- and DsRed-expressing cells were mixed in a 1 to 1 ratio; pairs were assessed by dual whole-cell patch clamping 6–48 h later. For each combination listed on the x -axis, the mean and SE of the G j and number of pairs tested is shown. Using a Kruskal–Wallis test followed by Dunn's multiple comparison test, only Cx32/Cx30 and Cx47/Cx43 channels have G j that is significantly greater (* p

    Article Snippet: Each connexin was subcloned into the bicistronic vectors pIRES2-EGFP (Clontech, Mountain View, CA) and pIRES2-DsRed; the latter was generated by replacing the enhanced green fluorescent protein (EGFP) sequence of the former with PCR amplified coding sequence for monomeric DsRed (from pDsRed-N1; Clontech), using primers that added BstX I and Not I restriction sites to the 5′ and 3′ ends, respectively.

    Techniques: Functional Assay, Transfection, Plasmid Preparation, Expressing

    Gene inactivation by oxidative guanine lesions. ( A ) Dot density plots obtained by flow-cytometric analyses of the expression of DsRed-Monomer and green fluorescent protein (GFP) in HeLa cells at indicated times after co-transfection with equal amounts of pZA and pDsRed plasmids. The plasmid encoding for GFP was either not damaged (oG = 0) or contained on average three oxidized guanines (oG = 3). The transfected cells that do not express GFP localize in the upper left quadrants (marked with asterisks). ( B ) Quantitative analyses of the data presented in A representing percentages of the transfected cells located in the upper left quadrants (left) and median GFP fluorescence in the cells with a high DsRed-Monomer expression level (right). ( C ) Detection of oxidized guanine bases (oG) in the damaged plasmids by incubation with Fpg DNA glycosylase. The numbers of oxidized guanines were determined from the ratios of covalently closed circular (ccc) to open circular (oc) forms as described in ‘Materials and Methods’ section.

    Journal: Nucleic Acids Research

    Article Title: Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region

    doi: 10.1093/nar/gkq170

    Figure Lengend Snippet: Gene inactivation by oxidative guanine lesions. ( A ) Dot density plots obtained by flow-cytometric analyses of the expression of DsRed-Monomer and green fluorescent protein (GFP) in HeLa cells at indicated times after co-transfection with equal amounts of pZA and pDsRed plasmids. The plasmid encoding for GFP was either not damaged (oG = 0) or contained on average three oxidized guanines (oG = 3). The transfected cells that do not express GFP localize in the upper left quadrants (marked with asterisks). ( B ) Quantitative analyses of the data presented in A representing percentages of the transfected cells located in the upper left quadrants (left) and median GFP fluorescence in the cells with a high DsRed-Monomer expression level (right). ( C ) Detection of oxidized guanine bases (oG) in the damaged plasmids by incubation with Fpg DNA glycosylase. The numbers of oxidized guanines were determined from the ratios of covalently closed circular (ccc) to open circular (oc) forms as described in ‘Materials and Methods’ section.

    Article Snippet: Plasmids and DNA damage conditions pDsRed-Monomer-N1 (pDsRed) plasmid was from Clontech (Saint-Germain-en-Laye, France). pEGFP-mODC-ZA (pZA) that encodes for enhanced green fluorescent protein has been described previously ( ).

    Techniques: Flow Cytometry, Expressing, Cotransfection, Plasmid Preparation, Transfection, Fluorescence, Incubation, Countercurrent Chromatography

    Quantitative comparison of the chromatin components in the undamaged and damaged plasmid DNA. ( A ) Experimental flowchart of transfection and chromatin preparation. The reference non-damaged pDsRed plasmid is introduced as an internal standard for chromatin immunoprecipitation. ( B ) Summary of DNA fragments analysed by real-time PCR. ( C ) DNA fragment size analyses after the reversal of cross-links in the samples transfected with an undamaged pZA (lane 1) or pZA containing three oxidized guanines (lane 2). M. molecular size marker. ( D ) ChIP enrichments of the undamaged pZA (oG = 0, white bars) and damaged pZA (oG = 3, black bars) DNA fragments, relative to the promoter fragment of the reference pDsRed plasmid (not plotted, equals to 1 in all graphs). Error bars indicate standard deviation of the mean for n ChIP experimens.

    Journal: Nucleic Acids Research

    Article Title: Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region

    doi: 10.1093/nar/gkq170

    Figure Lengend Snippet: Quantitative comparison of the chromatin components in the undamaged and damaged plasmid DNA. ( A ) Experimental flowchart of transfection and chromatin preparation. The reference non-damaged pDsRed plasmid is introduced as an internal standard for chromatin immunoprecipitation. ( B ) Summary of DNA fragments analysed by real-time PCR. ( C ) DNA fragment size analyses after the reversal of cross-links in the samples transfected with an undamaged pZA (lane 1) or pZA containing three oxidized guanines (lane 2). M. molecular size marker. ( D ) ChIP enrichments of the undamaged pZA (oG = 0, white bars) and damaged pZA (oG = 3, black bars) DNA fragments, relative to the promoter fragment of the reference pDsRed plasmid (not plotted, equals to 1 in all graphs). Error bars indicate standard deviation of the mean for n ChIP experimens.

    Article Snippet: Plasmids and DNA damage conditions pDsRed-Monomer-N1 (pDsRed) plasmid was from Clontech (Saint-Germain-en-Laye, France). pEGFP-mODC-ZA (pZA) that encodes for enhanced green fluorescent protein has been described previously ( ).

    Techniques: Plasmid Preparation, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Marker, Standard Deviation

    Association of the transfected plasmid with chromatin. Nuclei isolated from HeLa cells 24 h after transfection with undamaged pZA were mixed with pure pDsRed plasmid and treated with MNase as described in ‘Materials and Methods’ section. (Top) Agarose gel electrophoresis of the fractions obtained by isopycnic centrifugation of the MNase-treated nuclei and of the products of PCR performed with diluted fractions of the same CsCl gradient (numbers indicate the gradient fractions starting from the bottom). M, molecular size marker; nt, no template; p, plasmid DNA. (Bottom) Distribution of the two plasmids (transfected and naked) between the gradient fractions.

    Journal: Nucleic Acids Research

    Article Title: Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region

    doi: 10.1093/nar/gkq170

    Figure Lengend Snippet: Association of the transfected plasmid with chromatin. Nuclei isolated from HeLa cells 24 h after transfection with undamaged pZA were mixed with pure pDsRed plasmid and treated with MNase as described in ‘Materials and Methods’ section. (Top) Agarose gel electrophoresis of the fractions obtained by isopycnic centrifugation of the MNase-treated nuclei and of the products of PCR performed with diluted fractions of the same CsCl gradient (numbers indicate the gradient fractions starting from the bottom). M, molecular size marker; nt, no template; p, plasmid DNA. (Bottom) Distribution of the two plasmids (transfected and naked) between the gradient fractions.

    Article Snippet: Plasmids and DNA damage conditions pDsRed-Monomer-N1 (pDsRed) plasmid was from Clontech (Saint-Germain-en-Laye, France). pEGFP-mODC-ZA (pZA) that encodes for enhanced green fluorescent protein has been described previously ( ).

    Techniques: Transfection, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Centrifugation, Polymerase Chain Reaction, Marker

    Plasmid survival and reporter gene expression in HeLa cells. The cells were transfected with 400 ng pDsRed plasmid (non-damaged reference) together with the indicated amounts of pZA plasmids (non-damaged or damaged, as specified). ( A ) Relative GFP fluorescence as a function of the amounts of non-damaged pZA plasmid used for transfection. Means of three independent experiments (±SD). ( B ) Comparison of DNA amounts recovered from the cells (open columns) and GFP protein expression (black columns) for the range of transfection inputs of the non-damaged (oG = 0) and damaged (oG = 3) pZA plasmid. All values are expressed relative to the values obtained after transfection with 400 ng of undamaged pZA plasmid. ( C ) Messenger RNA expression 24 h after transfections with non-damaged (oG = 0) and damaged (oG = 3) pZA plasmids. cDNA copy numbers (mean and data range) were determined by real-time PCR relative to the copy numbers of DNA recovered from the transfected cells. Mean GAPDH cDNA copy number is used for normalisation between the samples (duplicates).

    Journal: Nucleic Acids Research

    Article Title: Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region

    doi: 10.1093/nar/gkq170

    Figure Lengend Snippet: Plasmid survival and reporter gene expression in HeLa cells. The cells were transfected with 400 ng pDsRed plasmid (non-damaged reference) together with the indicated amounts of pZA plasmids (non-damaged or damaged, as specified). ( A ) Relative GFP fluorescence as a function of the amounts of non-damaged pZA plasmid used for transfection. Means of three independent experiments (±SD). ( B ) Comparison of DNA amounts recovered from the cells (open columns) and GFP protein expression (black columns) for the range of transfection inputs of the non-damaged (oG = 0) and damaged (oG = 3) pZA plasmid. All values are expressed relative to the values obtained after transfection with 400 ng of undamaged pZA plasmid. ( C ) Messenger RNA expression 24 h after transfections with non-damaged (oG = 0) and damaged (oG = 3) pZA plasmids. cDNA copy numbers (mean and data range) were determined by real-time PCR relative to the copy numbers of DNA recovered from the transfected cells. Mean GAPDH cDNA copy number is used for normalisation between the samples (duplicates).

    Article Snippet: Plasmids and DNA damage conditions pDsRed-Monomer-N1 (pDsRed) plasmid was from Clontech (Saint-Germain-en-Laye, France). pEGFP-mODC-ZA (pZA) that encodes for enhanced green fluorescent protein has been described previously ( ).

    Techniques: Plasmid Preparation, Expressing, Transfection, Fluorescence, RNA Expression, Real-time Polymerase Chain Reaction

    QD655 can be used in combination with fluorescent proteins. (A-B). DsRed2 (green) and QD655 (red) emission spectra under an 800nm NLO (A) or 1050nm OPO (B) excitation. (C). Spectrally unmixed image of GL261 DsRed2-expressing cells (green) growing on a

    Journal: Biomedical Optics Express

    Article Title: Combination of an optical parametric oscillator and quantum-dots 655 to improve imaging depth of vasculature by intravital multicolor two-photon microscopy

    doi: 10.1364/BOE.7.002362

    Figure Lengend Snippet: QD655 can be used in combination with fluorescent proteins. (A-B). DsRed2 (green) and QD655 (red) emission spectra under an 800nm NLO (A) or 1050nm OPO (B) excitation. (C). Spectrally unmixed image of GL261 DsRed2-expressing cells (green) growing on a

    Article Snippet: GL261 murine glioma cells expressing DsRed2 (plasmid: pDsRed2-N1, Clontech,Mountain View, USA) were cultured as previously described [ , ] on glass-bottom Petri dishes.

    Techniques: Expressing

    GH attenuates endogenous NHEJ DNA repair by inhibiting DNA-PKcs phosphorylation. ( A ) hNCC containing a chromosomally integrated NHEJ reporter cassette were cotransfected with I-SceI and DsRed expression vectors and treated with 500 ng/ml GH overnight. The intact reporters are negative for GFP. Upon induction of a DSB by I-SceI digestion, the functional GFP gene is reconstituted. Cells were also transfected with pDsRed2-N1 as transfection control, and the percent of DsRed + cells indicates transfection efficiency. Cells were analyzed by FACS on day 5 after GH treatment, and the relative efficiency of DNA DSB repair was calculated as the ratio of GFP + cells/DsRed + cells. Representative analysis is shown. ( B ) Graph shows fold change in GFP positivity ± SEM in 5 independent assays. Controls represent cells not treated with GH. Differences were assessed with Tukey-adjusted mixed model regression. ( C .

    Journal: JCI Insight

    Article Title: Excess growth hormone suppresses DNA damage repair in epithelial cells

    doi: 10.1172/jci.insight.125762

    Figure Lengend Snippet: GH attenuates endogenous NHEJ DNA repair by inhibiting DNA-PKcs phosphorylation. ( A ) hNCC containing a chromosomally integrated NHEJ reporter cassette were cotransfected with I-SceI and DsRed expression vectors and treated with 500 ng/ml GH overnight. The intact reporters are negative for GFP. Upon induction of a DSB by I-SceI digestion, the functional GFP gene is reconstituted. Cells were also transfected with pDsRed2-N1 as transfection control, and the percent of DsRed + cells indicates transfection efficiency. Cells were analyzed by FACS on day 5 after GH treatment, and the relative efficiency of DNA DSB repair was calculated as the ratio of GFP + cells/DsRed + cells. Representative analysis is shown. ( B ) Graph shows fold change in GFP positivity ± SEM in 5 independent assays. Controls represent cells not treated with GH. Differences were assessed with Tukey-adjusted mixed model regression. ( C .

    Article Snippet: Stably transfected cells were selected for 10 days with 1 mg/ml of G418 and then cotransfected (by nucleofection) with 5 μg plasmid encoding I-SceI endonuclease (pCBASceI; Addgene, plasmid no. 26477) to induce DSBs, and 0.5 μg plasmid encoding DsRed (pDsRed2-N1; Clontech) to control for transfection efficiency.

    Techniques: Non-Homologous End Joining, Expressing, Functional Assay, Transfection, FACS