plasmid dnas Search Results


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  • 94
    Thermo Fisher plasmids pbr322 dna
    ( A ) Scanning image of intercalated <t>pBR322</t> <t>DNA</t> fragments adsorbed on PLL-coated glass slides (25 × 25 µm 2 , pH 6, vertical scanning direction). Brightness of the molecules measured in PE. Adsorbed molecules cover a larger area than diffusing ones (small lines in the upper section of the image). ( B ) Histogram of the brightness of the adsorbed pBR322 DNA fragments, background of 12 PE substracted. Bin width 2 PE. Histogram was fitted with Gaussian curve (black line). The reduced χ 2 of the fit is 0.75.
    Plasmids Pbr322 Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore puc19 dna plasmid
    Quantitative analysis of the thiazacridine and imidazacridine derivatives effects on the relaxation of <t>pUC19</t> <t>DNA</t> plasmid by human topoisomerase 1.
    Puc19 Dna Plasmid, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Sage Science episomal plasmid dna a bluepippin device
    Quantitative analysis of the thiazacridine and imidazacridine derivatives effects on the relaxation of <t>pUC19</t> <t>DNA</t> plasmid by human topoisomerase 1.
    Episomal Plasmid Dna A Bluepippin Device, supplied by Sage Science, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore plasmid dnas
    Quantitative analysis of the thiazacridine and imidazacridine derivatives effects on the relaxation of <t>pUC19</t> <t>DNA</t> plasmid by human topoisomerase 1.
    Plasmid Dnas, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pbr322 plasmid
    Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) <t>pBR322</t> plasmid <t>DNA.</t> Topoisomerase IIα (Topo IIα) was present
    Pbr322 Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa plasmid dnas
    Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) <t>pBR322</t> plasmid <t>DNA.</t> Topoisomerase IIα (Topo IIα) was present
    Plasmid Dnas, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher plasmid dnas
    Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) <t>pBR322</t> plasmid <t>DNA.</t> Topoisomerase IIα (Topo IIα) was present
    Plasmid Dnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 4384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher plasmid prset a plasmid dna
    Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) <t>pBR322</t> plasmid <t>DNA.</t> Topoisomerase IIα (Topo IIα) was present
    Plasmid Prset A Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher episomal dna replication
    Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) <t>pBR322</t> plasmid <t>DNA.</t> Topoisomerase IIα (Topo IIα) was present
    Episomal Dna Replication, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore plasmid dna expression plasmids
    Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) <t>pBR322</t> plasmid <t>DNA.</t> Topoisomerase IIα (Topo IIα) was present
    Plasmid Dna Expression Plasmids, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher dna plasmids
    Humoral immune responses in <t>p24CE</t> <t>DNA</t> vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).
    Dna Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2403 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore dna plasmid encoding cas9
    Humoral immune responses in <t>p24CE</t> <t>DNA</t> vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).
    Dna Plasmid Encoding Cas9, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology dna plasmids
    Humoral immune responses in <t>p24CE</t> <t>DNA</t> vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).
    Dna Plasmids, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc plasmids dna
    Genetic engineering of <t>HepG2</t> cells using TALENs. ( A ) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted <t>DNA</t> base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). * p
    Plasmids Dna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sangon Biotech dna plasmids
    Genetic engineering of <t>HepG2</t> cells using TALENs. ( A ) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted <t>DNA</t> base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). * p
    Dna Plasmids, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Qiagen plasmid dnas
    Genetic engineering of <t>HepG2</t> cells using TALENs. ( A ) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted <t>DNA</t> base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). * p
    Plasmid Dnas, supplied by Qiagen, used in various techniques. Bioz Stars score: 94/100, based on 4485 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen plasmid plus dna kits
    Genetic engineering of <t>HepG2</t> cells using TALENs. ( A ) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted <t>DNA</t> base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). * p
    Plasmid Plus Dna Kits, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) Scanning image of intercalated pBR322 DNA fragments adsorbed on PLL-coated glass slides (25 × 25 µm 2 , pH 6, vertical scanning direction). Brightness of the molecules measured in PE. Adsorbed molecules cover a larger area than diffusing ones (small lines in the upper section of the image). ( B ) Histogram of the brightness of the adsorbed pBR322 DNA fragments, background of 12 PE substracted. Bin width 2 PE. Histogram was fitted with Gaussian curve (black line). The reduced χ 2 of the fit is 0.75.

    Journal: Nucleic Acids Research

    Article Title: Sizing of single fluorescently stained DNA fragments by scanning microscopy

    doi: 10.1093/nar/gng138

    Figure Lengend Snippet: ( A ) Scanning image of intercalated pBR322 DNA fragments adsorbed on PLL-coated glass slides (25 × 25 µm 2 , pH 6, vertical scanning direction). Brightness of the molecules measured in PE. Adsorbed molecules cover a larger area than diffusing ones (small lines in the upper section of the image). ( B ) Histogram of the brightness of the adsorbed pBR322 DNA fragments, background of 12 PE substracted. Bin width 2 PE. Histogram was fitted with Gaussian curve (black line). The reduced χ 2 of the fit is 0.75.

    Article Snippet: 14 200 bp plasmid DNA was isolated from EcoRI cells with the Plasmid Mini Kit (Qiagen, Germany). m13mp18 RF1 DNA (7250 bp), the plasmids pBR322 DNA (4361 bp) and pUC19 DNA (2686 bp) were purchased from MBI Fermentas, Germany. m13mp18 RF1 DNA was digested at 36°C for 4 h by the restriction enzymes EcoRI and PagI (MBI Fermentas, Germany) into 2318 and 4932 bp fragments.

    Techniques:

    Gel electrophoresis of used DNA fragments (1) and an EcoRI and PagI digest of m13mp18 RF1 DNA (2), as described in Materials and Methods. ( 1 ) Lines: (a) 1985 bp linear DNA, (b) pUC19 plasmid DNA, (c) 4120 bp linear DNA, (d) pBR322 plasmid DNA, (e) m13mp18 RF1 DNA, (f) 14 000 bp plasmid DNA and (g) DNA molecular weight marker II [23 130, 9416, 6557, 4361, 2322, 2027 bp (Roche, Switzerland)]. ( 2 ) Lines: (a) m13mp18 RF1 DNA, (b) EcoRI/PagI digest of m13mp18 RF1 DNA, (c) DNA molecular weight marker II (Roche, Switzerland).

    Journal: Nucleic Acids Research

    Article Title: Sizing of single fluorescently stained DNA fragments by scanning microscopy

    doi: 10.1093/nar/gng138

    Figure Lengend Snippet: Gel electrophoresis of used DNA fragments (1) and an EcoRI and PagI digest of m13mp18 RF1 DNA (2), as described in Materials and Methods. ( 1 ) Lines: (a) 1985 bp linear DNA, (b) pUC19 plasmid DNA, (c) 4120 bp linear DNA, (d) pBR322 plasmid DNA, (e) m13mp18 RF1 DNA, (f) 14 000 bp plasmid DNA and (g) DNA molecular weight marker II [23 130, 9416, 6557, 4361, 2322, 2027 bp (Roche, Switzerland)]. ( 2 ) Lines: (a) m13mp18 RF1 DNA, (b) EcoRI/PagI digest of m13mp18 RF1 DNA, (c) DNA molecular weight marker II (Roche, Switzerland).

    Article Snippet: 14 200 bp plasmid DNA was isolated from EcoRI cells with the Plasmid Mini Kit (Qiagen, Germany). m13mp18 RF1 DNA (7250 bp), the plasmids pBR322 DNA (4361 bp) and pUC19 DNA (2686 bp) were purchased from MBI Fermentas, Germany. m13mp18 RF1 DNA was digested at 36°C for 4 h by the restriction enzymes EcoRI and PagI (MBI Fermentas, Germany) into 2318 and 4932 bp fragments.

    Techniques: Nucleic Acid Electrophoresis, Plasmid Preparation, Molecular Weight, Marker

    ( a ) Agarose gel electrophoresis of pBR 322 DNA (45 μM) after incubation with Ru6L at various +/− ratios in aqueous solution; ( b ) Hydrodynamic diameter and ( c ) zeta potential of the pBR 322 DNA (1.5 μM) incubation with Ru6L at various +/− ratios in an aqueous solution,as determined by DLS.

    Journal: Scientific Reports

    Article Title: A dendritic nano-sized hexanuclear ruthenium(II) complex as a one- and two-photon luminescent tracking non-viral gene vector

    doi: 10.1038/srep10707

    Figure Lengend Snippet: ( a ) Agarose gel electrophoresis of pBR 322 DNA (45 μM) after incubation with Ru6L at various +/− ratios in aqueous solution; ( b ) Hydrodynamic diameter and ( c ) zeta potential of the pBR 322 DNA (1.5 μM) incubation with Ru6L at various +/− ratios in an aqueous solution,as determined by DLS.

    Article Snippet: The plasmid pBR 322 DNA was obtained from MBI Fermentas, the plasmid pEGFP DNA was purchased from Clontech, and the plasmid pGL3 control vector and luciferase kid were obtained from Promega.

    Techniques: Agarose Gel Electrophoresis, Incubation

    Cs-TopIB supercoiled DNA relaxation assays over broad temperature range. ( A ) Effect of the temperature: assays were carried out for 2 min using 200 ng of pBR322 as a substrate of negative supercoiled DNA and 50 nM of Cs-TopIB protein at the indicated temperatures (°C). ( B ) Time courses assays were performed using 200 ng of pBR322 as a substrate of negative supercoiled DNA and 50 nM of Cs-TopIB protein at the indicated times (s). In both panel, Sc−: negatively supercoiled DNA; R: relaxed DNA; C: control reaction without enzyme.

    Journal: Nucleic Acids Research

    Article Title: topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB

    doi: 10.1093/nar/gkw097

    Figure Lengend Snippet: Cs-TopIB supercoiled DNA relaxation assays over broad temperature range. ( A ) Effect of the temperature: assays were carried out for 2 min using 200 ng of pBR322 as a substrate of negative supercoiled DNA and 50 nM of Cs-TopIB protein at the indicated temperatures (°C). ( B ) Time courses assays were performed using 200 ng of pBR322 as a substrate of negative supercoiled DNA and 50 nM of Cs-TopIB protein at the indicated times (s). In both panel, Sc−: negatively supercoiled DNA; R: relaxed DNA; C: control reaction without enzyme.

    Article Snippet: Plasmids and reagents Negatively supercoiled plasmid pBR322 DNA was purchased from Fermentas Life Science and kinetoplast DNA (kDNA) from Topogen.

    Techniques:

    Cs-TopIB thermoresistance, relaxation of both positively and negatively supercoiled DNA and activity in the absence of magnesium. ( A ) Cs-TopIB was pre-heated at the indicated temperatures for 20 min, then assayed for pBR322 relaxation for 20 min at 65°C using 200 ng of pBR322 and 130 nM of Cs-TopIB protein. ( B ) Assays on negatively and positively supercoiled DNA: negatively (SC−) and positively (SC+) supercoiled pBR322 were assayed for relaxation with Escherichia coli TopIA and Cs-TopIB enzymes at 30°C for 30 min in their respective reaction buffers (see panel C and Supplementary Figure S4 for time-course experiments). ( C ) Assays were carried out for 30 min at 30°C with 0.8 nM of Hs-Top1, 2 nM of Cs-TopIB and 6.8 nM of Hs-Top1mt and 200 ng of pBR322 as a negatively supercoiled substrate. Mg 2+ was added at the indicated final concentrations. ‘C’: reaction without enzyme. In all panels, SC−: negatively supercoiled DNA; SC+: positively supercoiled DNA; R: relaxed DNA.

    Journal: Nucleic Acids Research

    Article Title: topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB

    doi: 10.1093/nar/gkw097

    Figure Lengend Snippet: Cs-TopIB thermoresistance, relaxation of both positively and negatively supercoiled DNA and activity in the absence of magnesium. ( A ) Cs-TopIB was pre-heated at the indicated temperatures for 20 min, then assayed for pBR322 relaxation for 20 min at 65°C using 200 ng of pBR322 and 130 nM of Cs-TopIB protein. ( B ) Assays on negatively and positively supercoiled DNA: negatively (SC−) and positively (SC+) supercoiled pBR322 were assayed for relaxation with Escherichia coli TopIA and Cs-TopIB enzymes at 30°C for 30 min in their respective reaction buffers (see panel C and Supplementary Figure S4 for time-course experiments). ( C ) Assays were carried out for 30 min at 30°C with 0.8 nM of Hs-Top1, 2 nM of Cs-TopIB and 6.8 nM of Hs-Top1mt and 200 ng of pBR322 as a negatively supercoiled substrate. Mg 2+ was added at the indicated final concentrations. ‘C’: reaction without enzyme. In all panels, SC−: negatively supercoiled DNA; SC+: positively supercoiled DNA; R: relaxed DNA.

    Article Snippet: Plasmids and reagents Negatively supercoiled plasmid pBR322 DNA was purchased from Fermentas Life Science and kinetoplast DNA (kDNA) from Topogen.

    Techniques: Activity Assay

    CPT resistance of Cs-TopIB on DNA relaxation. ( A ) Time courses assays were carried out from 1 to 30 min at 30°C in the presence of 5% DMSO or 50 mM of CPT and 200 ng of a negatively supercoiled pBR322. ( B ) Time courses assays were carried out from 1 to 30 min at 30°C in the presence of 5% DMSO or 50 mM of CPT and 200 ng of a positively supercoiled pBR322. Assays in (A) or (B) were done with addition of 10, 6 and 5 nM of Cs-TopIB, Hs-Top1mt and Hs-Top1 respectively. SC−: negatively supercoiled DNA; SC+: positively supercoiled DNA; R: relaxed DNA; C:control reaction without enzyme. Asterisk represents the open circular form in the control and a mix of the open form and the entirely relaxed form of the plasmids for the assays with enzymes.

    Journal: Nucleic Acids Research

    Article Title: topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB

    doi: 10.1093/nar/gkw097

    Figure Lengend Snippet: CPT resistance of Cs-TopIB on DNA relaxation. ( A ) Time courses assays were carried out from 1 to 30 min at 30°C in the presence of 5% DMSO or 50 mM of CPT and 200 ng of a negatively supercoiled pBR322. ( B ) Time courses assays were carried out from 1 to 30 min at 30°C in the presence of 5% DMSO or 50 mM of CPT and 200 ng of a positively supercoiled pBR322. Assays in (A) or (B) were done with addition of 10, 6 and 5 nM of Cs-TopIB, Hs-Top1mt and Hs-Top1 respectively. SC−: negatively supercoiled DNA; SC+: positively supercoiled DNA; R: relaxed DNA; C:control reaction without enzyme. Asterisk represents the open circular form in the control and a mix of the open form and the entirely relaxed form of the plasmids for the assays with enzymes.

    Article Snippet: Plasmids and reagents Negatively supercoiled plasmid pBR322 DNA was purchased from Fermentas Life Science and kinetoplast DNA (kDNA) from Topogen.

    Techniques: Cycling Probe Technology

    Quantitative analysis of the thiazacridine and imidazacridine derivatives effects on the relaxation of pUC19 DNA plasmid by human topoisomerase 1.

    Journal: Molecules

    Article Title: Synthesis, DNA Binding and Topoisomerase I Inhibition Activity of Thiazacridine and Imidazacridine Derivatives

    doi: 10.3390/molecules181215035

    Figure Lengend Snippet: Quantitative analysis of the thiazacridine and imidazacridine derivatives effects on the relaxation of pUC19 DNA plasmid by human topoisomerase 1.

    Article Snippet: DNA Topoisomerase I Inhibition Assay Human topoisomerase I inhibition activity was determined using 100 ng of pUC19 DNA plasmid (from Sigma) and 1.0 unit of recombinant human (TOP1 7150) DNA topoisomerase I (Sigma-Aldrich) in relaxation buffer (0.01 M Tris-HCl, pH 7.5, 0.05 M KCl, 5 mM MgCl2 , 0.1 mM Na2 EDTA, 0.01% bovine serum albumin) after incubation with or without derivatives for 45 min at 37 °C.

    Techniques: Plasmid Preparation

    Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) pBR322 plasmid DNA. Topoisomerase IIα (Topo IIα) was present

    Journal: Journal of inorganic biochemistry

    Article Title: Cadmium is a Catalytic Inhibitor of DNA Topoisomerase II

    doi: 10.1016/j.jinorgbio.2011.02.007

    Figure Lengend Snippet: Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) pBR322 plasmid DNA. Topoisomerase IIα (Topo IIα) was present

    Article Snippet: The 20 μL cleavage assay reaction mixture contained either 150 ng of topoisomerase IIα protein as indicated, 50 ng closed circular plasmid pBR322 plasmid DNA (MBI Fermentas, Burlington, Canada), 0.5 mM ATP in assay buffer (10 mM Tris-HCl, 50 mM KCl, 50 mM NaCl, 0.1 mM EDTA, 5 mM MgCl2 , 2.5 μg/mL bovine serum albumin, 1.5 μM dithiothreitol from the topoisomerase IIα preparation, 2.5% (v/v) glycerol, pH 8.0) and cadmium chloride or etoposide as indicated.

    Techniques: Staining, Plasmid Preparation

    Humoral immune responses in p24CE DNA vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).

    Journal: PLoS ONE

    Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

    doi: 10.1371/journal.pone.0060245

    Figure Lengend Snippet: Humoral immune responses in p24CE DNA vaccinated mice. ( A ) Anti-HIV-1 p24 gag antibodies were measured in plasma from p24CE and p55 gag DNA vaccinated C57BL/6 mice by a standard clade B p24 gag ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55 gag DNA (bottom panel). ( B ) Humoral responses induced upon SP-p24CE or p55 gag DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24 gag protein collected from supernatants of HEK293 cells transfected with 5 µg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1∶5000 dilution) from mice vaccinated with a mixture of SP-p24CE1 2 DNAs (top panel) or p55 gag DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. ( C ) Detection of humoral responses to full-length p55 gag in mice vaccinated with p24CE or p55 gag DNA by Western immunoblot assay. The p55 gag proteins were obtained from HEK293 cells transfected with 0.5 µg of RNA/codon optimized plasmids expressing unprocessed p55 gag from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1∶200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55 gag (bottom panel).

    Article Snippet: DNA plasmids The p24CE and gag gene coding sequences were designed by RNA/codon optimization for efficient expression in mammalian cells – and chemically synthesized (GeneArt, Life Technologies, Grand Island, NY).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Labeling, Expressing

    Phenotypic and functional analysis of T cell responses generated by p55 gag and p24CE DNA vaccination. (A) Mice (N = 5/group) were vaccinated 3 times (week 0, 3 and 6) with 20 µg of a plasmid expressing HXB2 p55 gag (clade B) or 20 µg of a mixture of plasmids expressing SP-p24CE1 and SP-p24CE2. The mice were sacrificed 2 weeks after the last immunization. Three independent experiments were performed and a representative experiment is shown. ( B ) Pooled splenocytes were stimulated with Clade A, B or C peptide pools (15-mers) spanning the p24 gag region (left panel) and the Group M consensus peptide pool (right panel). The frequency of the CD4 + (open bars) and CD8 + (filled bars) p24 gag -specific IFN-γ producing T cells was determined. ( C ) The splenocytes from the SP-p24CE (left panel) and p55 gag (right panel) DNA vaccinated mice were stimulated with peptide pools specific for the individual CEs. The frequency of the CD4 + (open bars) and CD8 + (filled bars) CE-specific IFN-γ producing T cells was determined. ( D ) Plot overlays show the phenotypic and functional characterization of the antigen-specific T cells induced by SP-p24CE (left panels) and p55 gag (right panels) DNA vaccines upon stimulation with p24 gag –specific peptide pool. Total T cells recovered from the spleen are shown as grey contours, and the antigen-specific IFN-γ + T cells are overlaid as red (CD4 + T cells) or black (CD8 + T cells) dots. The plots show the CD4/CD8 distribution (top panel), memory phenotype as determined by CD44/CD62L staining (middle panel) and TNF-α/CD107a expression (bottom panel) among the T cells from vaccinated mice. The frequency of CD4 (red) and CD8 (black) IFN-γ T lymphocytes is shown.

    Journal: PLoS ONE

    Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

    doi: 10.1371/journal.pone.0060245

    Figure Lengend Snippet: Phenotypic and functional analysis of T cell responses generated by p55 gag and p24CE DNA vaccination. (A) Mice (N = 5/group) were vaccinated 3 times (week 0, 3 and 6) with 20 µg of a plasmid expressing HXB2 p55 gag (clade B) or 20 µg of a mixture of plasmids expressing SP-p24CE1 and SP-p24CE2. The mice were sacrificed 2 weeks after the last immunization. Three independent experiments were performed and a representative experiment is shown. ( B ) Pooled splenocytes were stimulated with Clade A, B or C peptide pools (15-mers) spanning the p24 gag region (left panel) and the Group M consensus peptide pool (right panel). The frequency of the CD4 + (open bars) and CD8 + (filled bars) p24 gag -specific IFN-γ producing T cells was determined. ( C ) The splenocytes from the SP-p24CE (left panel) and p55 gag (right panel) DNA vaccinated mice were stimulated with peptide pools specific for the individual CEs. The frequency of the CD4 + (open bars) and CD8 + (filled bars) CE-specific IFN-γ producing T cells was determined. ( D ) Plot overlays show the phenotypic and functional characterization of the antigen-specific T cells induced by SP-p24CE (left panels) and p55 gag (right panels) DNA vaccines upon stimulation with p24 gag –specific peptide pool. Total T cells recovered from the spleen are shown as grey contours, and the antigen-specific IFN-γ + T cells are overlaid as red (CD4 + T cells) or black (CD8 + T cells) dots. The plots show the CD4/CD8 distribution (top panel), memory phenotype as determined by CD44/CD62L staining (middle panel) and TNF-α/CD107a expression (bottom panel) among the T cells from vaccinated mice. The frequency of CD4 (red) and CD8 (black) IFN-γ T lymphocytes is shown.

    Article Snippet: DNA plasmids The p24CE and gag gene coding sequences were designed by RNA/codon optimization for efficient expression in mammalian cells – and chemically synthesized (GeneArt, Life Technologies, Grand Island, NY).

    Techniques: Functional Assay, Generated, Mouse Assay, Plasmid Preparation, Expressing, Staining

    Cellular responses in p24CE DNA vaccinated C57BL/6 mice. Mice were vaccinated using in vivo EP with 20 µg of the indicated p24CE1 (left panel) or p24CE2 (right panel) DNA plasmids ( A, B ) or with SP-p24CE1 DNA ( C ). Splenocytes from individual animals were stimulated ( A ) with the Group M Consensus peptide pool (15-mer peptides overlapping by 11 AA), ( B ) with the COT-M peptide pool (10-mers overlapping by 9 AA) consisting of the matching peptides of p24CE1 and p24CE2 proteins, and ( C ) with peptide pools representing the clade A, B, and C p55 gag sequences (15-mer peptides overlapping by 11 AA), as described in Materials and Methods . The frequency of CE-specific IFN-γ producing CD4 + (open bars) and CD8 + (filled bars) T cells was determined by polychromatic flow cytometry. The mean and SEM are shown. Three experiments were performed and data from a representative experiment are shown.

    Journal: PLoS ONE

    Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

    doi: 10.1371/journal.pone.0060245

    Figure Lengend Snippet: Cellular responses in p24CE DNA vaccinated C57BL/6 mice. Mice were vaccinated using in vivo EP with 20 µg of the indicated p24CE1 (left panel) or p24CE2 (right panel) DNA plasmids ( A, B ) or with SP-p24CE1 DNA ( C ). Splenocytes from individual animals were stimulated ( A ) with the Group M Consensus peptide pool (15-mer peptides overlapping by 11 AA), ( B ) with the COT-M peptide pool (10-mers overlapping by 9 AA) consisting of the matching peptides of p24CE1 and p24CE2 proteins, and ( C ) with peptide pools representing the clade A, B, and C p55 gag sequences (15-mer peptides overlapping by 11 AA), as described in Materials and Methods . The frequency of CE-specific IFN-γ producing CD4 + (open bars) and CD8 + (filled bars) T cells was determined by polychromatic flow cytometry. The mean and SEM are shown. Three experiments were performed and data from a representative experiment are shown.

    Article Snippet: DNA plasmids The p24CE and gag gene coding sequences were designed by RNA/codon optimization for efficient expression in mammalian cells – and chemically synthesized (GeneArt, Life Technologies, Grand Island, NY).

    Techniques: Mouse Assay, In Vivo, Flow Cytometry, Cytometry

    Expression of the p24CE plasmids upon transient transfection in cultured cells. Plasmid DNA (1 µg) expressing different variants of either p24CE1 (left panel) or p24CE2 (right panel) proteins were transfected in HEK293 cells. The cultures were harvested 24 hrs later and proteins from equal amounts (1/250) from the cell-associated (top panel) and extra-cellular (bottom panel) fractions were resolved on a 12% NuPAGE Bis-Tris gel and analyzed by Western immunoblot using a goat anti-p24 gag antiserum and visualized using enhanced ECL. The membrane containing the cell-associated fractions was also probed with anti-human pan actin antibody to control for equal loading of the samples.

    Journal: PLoS ONE

    Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

    doi: 10.1371/journal.pone.0060245

    Figure Lengend Snippet: Expression of the p24CE plasmids upon transient transfection in cultured cells. Plasmid DNA (1 µg) expressing different variants of either p24CE1 (left panel) or p24CE2 (right panel) proteins were transfected in HEK293 cells. The cultures were harvested 24 hrs later and proteins from equal amounts (1/250) from the cell-associated (top panel) and extra-cellular (bottom panel) fractions were resolved on a 12% NuPAGE Bis-Tris gel and analyzed by Western immunoblot using a goat anti-p24 gag antiserum and visualized using enhanced ECL. The membrane containing the cell-associated fractions was also probed with anti-human pan actin antibody to control for equal loading of the samples.

    Article Snippet: DNA plasmids The p24CE and gag gene coding sequences were designed by RNA/codon optimization for efficient expression in mammalian cells – and chemically synthesized (GeneArt, Life Technologies, Grand Island, NY).

    Techniques: Expressing, Transfection, Cell Culture, Plasmid Preparation, Western Blot

    Design of the p24CE DNA vaccine. ( A ) Alignment of the HXB2 p24 gag protein sequences with the consensus clades A, B and C and the Group M consensus, the Group M Center-of-Tree (COT-M) and the 7 CE included in p24CE1 and p24CE2. The ‘toggle’ amino acid differences between the CE1 and CE2 sequences are indicated. ( B ) Localization of CE within the hexameric p24 gag structure. The p24 gag structure is modified from Pornillos et al. [57] and shows the location of CE1-CE7 (red), the toggle AA (blue) and the AA not included in the CE (black). The crystal structure of the hexamer was obtained from http://www.ebi.ac.uk/pdbsum/ . ( C ) Kyte-Dolittle hydrophobicity plots for two different collinear arrangements of the CEs. ( D ) The p24CE (p24CE1 and p24CE2) proteins are composed of 7 CE arranged collinearly and linked via amino acid linkers. The secreted SP-p24CE contains the GM-CSF signal peptide. MCP3-p24CE is a fusion protein with the Monocyte chemoattractant protein 3 (MCP3) chemokine. LAMP-p24CE is a fusion with the lysosomal associated membrane protein 1 (LAMP-1).

    Journal: PLoS ONE

    Article Title: HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice

    doi: 10.1371/journal.pone.0060245

    Figure Lengend Snippet: Design of the p24CE DNA vaccine. ( A ) Alignment of the HXB2 p24 gag protein sequences with the consensus clades A, B and C and the Group M consensus, the Group M Center-of-Tree (COT-M) and the 7 CE included in p24CE1 and p24CE2. The ‘toggle’ amino acid differences between the CE1 and CE2 sequences are indicated. ( B ) Localization of CE within the hexameric p24 gag structure. The p24 gag structure is modified from Pornillos et al. [57] and shows the location of CE1-CE7 (red), the toggle AA (blue) and the AA not included in the CE (black). The crystal structure of the hexamer was obtained from http://www.ebi.ac.uk/pdbsum/ . ( C ) Kyte-Dolittle hydrophobicity plots for two different collinear arrangements of the CEs. ( D ) The p24CE (p24CE1 and p24CE2) proteins are composed of 7 CE arranged collinearly and linked via amino acid linkers. The secreted SP-p24CE contains the GM-CSF signal peptide. MCP3-p24CE is a fusion protein with the Monocyte chemoattractant protein 3 (MCP3) chemokine. LAMP-p24CE is a fusion with the lysosomal associated membrane protein 1 (LAMP-1).

    Article Snippet: DNA plasmids The p24CE and gag gene coding sequences were designed by RNA/codon optimization for efficient expression in mammalian cells – and chemically synthesized (GeneArt, Life Technologies, Grand Island, NY).

    Techniques: Modification

    Genetic engineering of HepG2 cells using TALENs. ( A ) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted DNA base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). * p

    Journal: Scientific Reports

    Article Title: Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

    doi: 10.1038/srep40446

    Figure Lengend Snippet: Genetic engineering of HepG2 cells using TALENs. ( A ) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted DNA base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). * p

    Article Snippet: Luciferase reporter assay To test whether hypoxia activated the WNT/β-catenin pathway, HepG2 and smmc-7721 cells were transfected with DNA plasmids of β-catenin-LEF/TCF-sensitive (TOP-flash) or β-catenin-LEF/TCF insensitive (FOP-flash) reporter vectors (Addgene, Cambridge, MA).

    Techniques: TALENs, Sequencing, Polymerase Chain Reaction, Construct, SSA Assay, Activity Assay, Genomic Sequencing, Clone Assay, Cell Culture, Western Blot

    Hypoxia transactivates hypoxia-responsive elements (HREs) in the BCL9 promoter which transcriptionally regulated by HIF-1α. ( A ) The human BCL9 gene contains 3 putative HREs in its promoter region. ( B ) Hypoxia activates the luciferase activity of reporter vectors containing HRE-B or HRE-C sites in the BCL9 promoter. HepG2 and SMMC-7721 cells were transfected with the luciferase reporter vectors, and then subjected to hypoxia treatment for 36 h before measuring luciferase activities. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. ( C,D ) HIF-1α but not HIF-2α binds to HRE-B and HRE-C sites in the BCL9 promoter under the hypoxic condition in HepG2 cells as determined by ChIP assays. Cells were cultured under the hypoxic or normoxic conditions for 36 h before assays. The HRE site in the VEGF promoter serves as a positive control. The amount of DNA fragments pulled- down was determined by real-time PCR ( C ) or conventional PCR ( D ). ( E ) Ectopic HIF-1α expression increases BCL9 protein levels in HepG2 cells as determined by Western-blot assays. ( F,G ) HIF-1α binds to HRE-B and HRE-C sites in the BCL9 promoter in HepG2 cells transfected with HIF-1α expression plasmids as determined by ChIP assays. The amount of DNA fragments pulled-down was determined by real-time PCR ( F ) or conventional PCR ( F ). The HRE site in the VEGF promoter serves as a positive control. Data are presented as mean ± SD (n = 3). * p

    Journal: Scientific Reports

    Article Title: Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

    doi: 10.1038/srep40446

    Figure Lengend Snippet: Hypoxia transactivates hypoxia-responsive elements (HREs) in the BCL9 promoter which transcriptionally regulated by HIF-1α. ( A ) The human BCL9 gene contains 3 putative HREs in its promoter region. ( B ) Hypoxia activates the luciferase activity of reporter vectors containing HRE-B or HRE-C sites in the BCL9 promoter. HepG2 and SMMC-7721 cells were transfected with the luciferase reporter vectors, and then subjected to hypoxia treatment for 36 h before measuring luciferase activities. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. ( C,D ) HIF-1α but not HIF-2α binds to HRE-B and HRE-C sites in the BCL9 promoter under the hypoxic condition in HepG2 cells as determined by ChIP assays. Cells were cultured under the hypoxic or normoxic conditions for 36 h before assays. The HRE site in the VEGF promoter serves as a positive control. The amount of DNA fragments pulled- down was determined by real-time PCR ( C ) or conventional PCR ( D ). ( E ) Ectopic HIF-1α expression increases BCL9 protein levels in HepG2 cells as determined by Western-blot assays. ( F,G ) HIF-1α binds to HRE-B and HRE-C sites in the BCL9 promoter in HepG2 cells transfected with HIF-1α expression plasmids as determined by ChIP assays. The amount of DNA fragments pulled-down was determined by real-time PCR ( F ) or conventional PCR ( F ). The HRE site in the VEGF promoter serves as a positive control. Data are presented as mean ± SD (n = 3). * p

    Article Snippet: Luciferase reporter assay To test whether hypoxia activated the WNT/β-catenin pathway, HepG2 and smmc-7721 cells were transfected with DNA plasmids of β-catenin-LEF/TCF-sensitive (TOP-flash) or β-catenin-LEF/TCF insensitive (FOP-flash) reporter vectors (Addgene, Cambridge, MA).

    Techniques: Luciferase, Activity Assay, Transfection, Positive Control, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing, Western Blot