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  • 99
    Integrated DNA Technologies cas9 expressing plasmid
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Cas9 Expressing Plasmid, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 8 article reviews
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    99
    Millipore plasmids expressing cas9
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Plasmids Expressing Cas9, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Addgene inc expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 1100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8097 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BestGene Inc expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by BestGene Inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    OriGene expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by OriGene, used in various techniques. Bioz Stars score: 99/100, based on 393 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1627 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    SABiosciences expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by SABiosciences, used in various techniques. Bioz Stars score: 86/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 97/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa expression plasmids
    Phenotypic manifestations of RDEB in <t>CRISPR/Cas9</t> knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.
    Expression Plasmids, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Vigene Biosciences csde1 expressing plasmid
    HeLa cells were transiently transfected with siRNAs and sgRNAs targeting ACBD3 or <t>CSDE1</t> . ( A ) Cells were infected with CVB3, fixed, and stained. The percentage of CVB3-infected cells (normalized to sgRNA or siRNA control). Mean ± SEM for triplicate experiments. (**) P
    Csde1 Expressing Plasmid, supplied by Vigene Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher epha7 expression plasmids
    HeLa cells were transiently transfected with siRNAs and sgRNAs targeting ACBD3 or <t>CSDE1</t> . ( A ) Cells were infected with CVB3, fixed, and stained. The percentage of CVB3-infected cells (normalized to sgRNA or siRNA control). Mean ± SEM for triplicate experiments. (**) P
    Epha7 Expression Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Addgene inc cas9 expressing plasmid
    One step mini-chromosome construction by CRISPR-PCS. ( a ) One step mini-chromosome construction using CRISPR-PCS. The chromosomal region between C12-P1 and C12-P2 of Chr. XII was targeted to form a mini-chromosome. A 400 bp homology sequence was used in the splitting modules. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. Left panel, PFGE analysis of wild type <t>FY834-Cas9</t> and 10 randomly chosen transformants. Right panel, Southern blot analysis after PFGE using probe 3 for detection of newly generated 31 kb mini-chromosome. The arrow beside the right panel represents the 31 kb expected mini-chromosome (lane 1, 3, 6, and 8). The 57 kb band in lane 2 and 7 show these strains have only one split at C12-P1. ( b ) One step mini-chromosome construction using CRISPR-PCS under the same experimental conditions as above except for use of a 50 bp homology sequence in the splitting module. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. The arrow beside the right panel represents the 31 kb expected mini-chromosome. The expected mini-chromosome was constructed in two strains (Lane 2 and 8). Four strains are shown to have single splitting at C12-P1 (Lane 3, 4, 6, and 9). Four strains are shown to have single splitting at C12-P2 (Lane 1, 5, 7, and 10). W. Wild type.
    Cas9 Expressing Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen hdac6 expression plasmids
    One step mini-chromosome construction by CRISPR-PCS. ( a ) One step mini-chromosome construction using CRISPR-PCS. The chromosomal region between C12-P1 and C12-P2 of Chr. XII was targeted to form a mini-chromosome. A 400 bp homology sequence was used in the splitting modules. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. Left panel, PFGE analysis of wild type <t>FY834-Cas9</t> and 10 randomly chosen transformants. Right panel, Southern blot analysis after PFGE using probe 3 for detection of newly generated 31 kb mini-chromosome. The arrow beside the right panel represents the 31 kb expected mini-chromosome (lane 1, 3, 6, and 8). The 57 kb band in lane 2 and 7 show these strains have only one split at C12-P1. ( b ) One step mini-chromosome construction using CRISPR-PCS under the same experimental conditions as above except for use of a 50 bp homology sequence in the splitting module. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. The arrow beside the right panel represents the 31 kb expected mini-chromosome. The expected mini-chromosome was constructed in two strains (Lane 2 and 8). Four strains are shown to have single splitting at C12-P1 (Lane 3, 4, 6, and 9). Four strains are shown to have single splitting at C12-P2 (Lane 1, 5, 7, and 10). W. Wild type.
    Hdac6 Expression Plasmids, supplied by InvivoGen, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Genecopoeia vdr expression plasmids
    1,25(OH) 2 D 2 inhibits LPS-induced COX-2 and phospho-AKT T308 in the <t>RAW264.</t> 7 cells. RAW264.7 cells were exposed to 250 n m 1,25(OH) 2 D 2 or <t>VDR</t> siRNAs ( si-VDR ), and cell lysates were analyzed to Western blotting. The experiments were repeated three times.
    Vdr Expression Plasmids, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Addgene inc plasmids expressing pbx1a
    1,25(OH) 2 D 2 inhibits LPS-induced COX-2 and phospho-AKT T308 in the <t>RAW264.</t> 7 cells. RAW264.7 cells were exposed to 250 n m 1,25(OH) 2 D 2 or <t>VDR</t> siRNAs ( si-VDR ), and cell lysates were analyzed to Western blotting. The experiments were repeated three times.
    Plasmids Expressing Pbx1a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Addgene inc jnk1a1 expressing plasmid
    1,25(OH) 2 D 2 inhibits LPS-induced COX-2 and phospho-AKT T308 in the <t>RAW264.</t> 7 cells. RAW264.7 cells were exposed to 250 n m 1,25(OH) 2 D 2 or <t>VDR</t> siRNAs ( si-VDR ), and cell lysates were analyzed to Western blotting. The experiments were repeated three times.
    Jnk1a1 Expressing Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore expression plasmids naphthalene
    1,25(OH) 2 D 2 inhibits LPS-induced COX-2 and phospho-AKT T308 in the <t>RAW264.</t> 7 cells. RAW264.7 cells were exposed to 250 n m 1,25(OH) 2 D 2 or <t>VDR</t> siRNAs ( si-VDR ), and cell lysates were analyzed to Western blotting. The experiments were repeated three times.
    Expression Plasmids Naphthalene, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc zebrafish expression plasmids
    1,25(OH) 2 D 2 inhibits LPS-induced COX-2 and phospho-AKT T308 in the <t>RAW264.</t> 7 cells. RAW264.7 cells were exposed to 250 n m 1,25(OH) 2 D 2 or <t>VDR</t> siRNAs ( si-VDR ), and cell lysates were analyzed to Western blotting. The experiments were repeated three times.
    Zebrafish Expression Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological gnl3 expression plasmids
    Effects of <t>GNL3</t> knockdown and overexpression on the levels of the EMT-related markers, β-catenin and GNL3 in vitro . (A and B) In HCT-116 cells, GNL3 knockdown (GNL3 shRNA) increased E-cadherin levels and reduced N-cadherin, vimentin, nuclear β-catenin (β-catenin-N) and GNL3 levels compared with controls (Con). (A and C) In HT-29 cells, GNL3 overexpression (GNL3) increased the N-cadherin, vimentin, β-catenin-N and GNL3 levels and reduced the E-cadherin levels compared with the negative controls (NC). **P
    Gnl3 Expression Plasmids, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Addgene inc hdac4 expression plasmids
    <t>HDAC4</t> is a direct target of miR-365. A ) Diagram of predicted miR-365 sequential pairing with a putative target region (miR-365 binding site) in the 3′-UTR of HDAC4 mRNA. The 5′ end of miR-365 contains a base pair sequence complementary
    Hdac4 Expression Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene il22ra2i3 expression plasmids
    <t>HDAC4</t> is a direct target of miR-365. A ) Diagram of predicted miR-365 sequential pairing with a putative target region (miR-365 binding site) in the 3′-UTR of HDAC4 mRNA. The 5′ end of miR-365 contains a base pair sequence complementary
    Il22ra2i3 Expression Plasmids, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    TaKaRa tta expressing plasmid
    <t>HDAC4</t> is a direct target of miR-365. A ) Diagram of predicted miR-365 sequential pairing with a putative target region (miR-365 binding site) in the 3′-UTR of HDAC4 mRNA. The 5′ end of miR-365 contains a base pair sequence complementary
    Tta Expressing Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega psvl expression plasmids
    <t>HDAC4</t> is a direct target of miR-365. A ) Diagram of predicted miR-365 sequential pairing with a putative target region (miR-365 binding site) in the 3′-UTR of HDAC4 mRNA. The 5′ end of miR-365 contains a base pair sequence complementary
    Psvl Expression Plasmids, supplied by Promega, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega pgl3 plasmid expressing
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    Pgl3 Plasmid Expressing, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    TaKaRa vsvg expressing plasmid
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    Vsvg Expressing Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore expression pet28a plasmid
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    Expression Pet28a Plasmid, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Arbor Vita e6 expression plasmids
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    E6 Expression Plasmids, supplied by Arbor Vita, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher cbp expressing plasmid
    HEK293T cells were transiently transfected with 80 ng of p(Gal) 2 -50.huIL6P-luc+ and with various expression plasmids, the total amount of DNA being fixed at 400 ng, i.e., <t>pGal4</t> (40 ng) or pGal4-p65 (10 ng) ( A ) or pGal4-VP16 (40 ng) ( B ), whether or not with <t>pCMV-CBP</t> (25 ng or 100 ng) and/or pSVhGRα (25 ng or 100 ng). In control lanes with CBP and GR, + corresponds to the highest concentration. Assays were performed in triplicate and are representative of two independent experiments.
    Cbp Expressing Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Phenotypic manifestations of RDEB in CRISPR/Cas9 knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Rapid generation of Col7a1−/− mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells

    doi: 10.1038/labinvest.2017.85

    Figure Lengend Snippet: Phenotypic manifestations of RDEB in CRISPR/Cas9 knockout NSG mice ( a ) Phenotypic manifestations of RDEB in Col7a1 −/− NSG mice. Neonatal mice exhibit blistered paws shortly after birth, followed by formation of the more severe blisters and open wounds characteristic of skin fragility. ( b ) Immunofluorescence staining of type VII collagen expression in Col7a1 −/− NSG mice. Cross-sections of skin and esophagus in wild-type and knockout neonates showing the absence of type VII (red) in the esophageal membrane and at the dermal-epidermal junction in skin. ( c ) Representative patterns of Col7a1 mutations produced by CRISPR/Cas9 nuclease activity after embryo injection. Indels are observed at both gRNA target sites independently and simultaneously. gRNA-spanning deletions are also observed.

    Article Snippet: For validation, gRNAs were cloned into a U6 expression vector and co-delivered with a Cas9-expressing plasmid into 3T3 cells followed by determination of nuclease activity by Surveyor assay (Integrated DNA Technologies, Coralville, IA).

    Techniques: CRISPR, Knock-Out, Mouse Assay, Immunofluorescence, Staining, Expressing, Produced, Activity Assay, Injection

    CRISPR/Cas9-based disruption of type VII collagen by embryo injection ( a ) Strategy using the CRISPR/Cas9 nuclease system to produce Col7a1 −/− NSG mice. CRISPR guide RNA and Cas9 mRNA are injected into cytoplasm of single-cell NSG embryos, which are then transferred to CD-1 pseudo-pregnant female surrogates. Upon birth, visibly blistered animals were used for transplantation and/or survival experiments while the non-blistered animals were kept for genotyping and subsequent breeding colony establishment. ( b ) First coding exon of murine Col7a1 . To maximize the frequency of frameshift mutations, two gRNAs (green text) were designed to cut near each other within first coding exon. Start codon in red, PAM sequences in orange. Cut site indicated by red triangle. ( c ) Surveyor assay from transient transfection used to validate the nuclease assay of each gRNA. Red triangles indicate the Surveyor cleavage products.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Rapid generation of Col7a1−/− mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells

    doi: 10.1038/labinvest.2017.85

    Figure Lengend Snippet: CRISPR/Cas9-based disruption of type VII collagen by embryo injection ( a ) Strategy using the CRISPR/Cas9 nuclease system to produce Col7a1 −/− NSG mice. CRISPR guide RNA and Cas9 mRNA are injected into cytoplasm of single-cell NSG embryos, which are then transferred to CD-1 pseudo-pregnant female surrogates. Upon birth, visibly blistered animals were used for transplantation and/or survival experiments while the non-blistered animals were kept for genotyping and subsequent breeding colony establishment. ( b ) First coding exon of murine Col7a1 . To maximize the frequency of frameshift mutations, two gRNAs (green text) were designed to cut near each other within first coding exon. Start codon in red, PAM sequences in orange. Cut site indicated by red triangle. ( c ) Surveyor assay from transient transfection used to validate the nuclease assay of each gRNA. Red triangles indicate the Surveyor cleavage products.

    Article Snippet: For validation, gRNAs were cloned into a U6 expression vector and co-delivered with a Cas9-expressing plasmid into 3T3 cells followed by determination of nuclease activity by Surveyor assay (Integrated DNA Technologies, Coralville, IA).

    Techniques: CRISPR, Injection, Mouse Assay, Transplantation Assay, Transfection, Nuclease Assay

    Immunomodulation alleviates RDEB pathology ( a ) Survival of Col7a1 −/− NSG (n=15) and immune-competent C57BL/6 (n=10) mice, created by CRISPR/Cas9 embryo injection ( P

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Rapid generation of Col7a1−/− mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells

    doi: 10.1038/labinvest.2017.85

    Figure Lengend Snippet: Immunomodulation alleviates RDEB pathology ( a ) Survival of Col7a1 −/− NSG (n=15) and immune-competent C57BL/6 (n=10) mice, created by CRISPR/Cas9 embryo injection ( P

    Article Snippet: For validation, gRNAs were cloned into a U6 expression vector and co-delivered with a Cas9-expressing plasmid into 3T3 cells followed by determination of nuclease activity by Surveyor assay (Integrated DNA Technologies, Coralville, IA).

    Techniques: Mouse Assay, CRISPR, Injection

    HeLa cells were transiently transfected with siRNAs and sgRNAs targeting ACBD3 or CSDE1 . ( A ) Cells were infected with CVB3, fixed, and stained. The percentage of CVB3-infected cells (normalized to sgRNA or siRNA control). Mean ± SEM for triplicate experiments. (**) P

    Journal: Genome Research

    Article Title: Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

    doi: 10.1101/gr.230250.117

    Figure Lengend Snippet: HeLa cells were transiently transfected with siRNAs and sgRNAs targeting ACBD3 or CSDE1 . ( A ) Cells were infected with CVB3, fixed, and stained. The percentage of CVB3-infected cells (normalized to sgRNA or siRNA control). Mean ± SEM for triplicate experiments. (**) P

    Article Snippet: A CSDE1-expressing plasmid was purchased form Vigene Bioscience ( ) and a plasmid encoding eGFP-CSDE1 was constructed using the eGFP-N1 plasmid (Clontech Laboratories). pCI-FLAG–ACBD3, which contains a FLAG-tagged ACBD3 gene, was obtained from Jun Sasaki (Fujita Health University, Aichi, Japan) ( ).

    Techniques: Transfection, Infection, Staining

    CSDE1 is required for CVB3 infection. ( A ) Viability of wild-type (HeLa) and CSDE1 knockout (KO) cells after CVB3 infection. ( B ) Viral replication test of wild-type (HeLa) and CSDE1 KO cells using the CVB3 replicon. Transfection with cDNA encoding the eGFP-CSDE1 protein into CSDE1 KO cells rescued CVB3 replication. ( C ) Transfection with cDNA encoding the eGFP-CSDE1 protein into CSDE1 KO cells rescued CVB3 infection. After 8 h of CVB3 infection in wild-type (HeLa) and CSDE1 KO cells transfected with the plasmid encoding the eGFP-CSDE1 protein, cells were harvested and lysed for western blot analysis using anti-CSDE1 antibody, anti-VP1 antibody, and anti-beta actin antibody. ( D ) CVB3 IRES-dependent translation assay of wild-type (HeLa) and CSDE1 KO cells. Dual luciferase reporter plasmids containing the IRES sequence from five different viruses were transfected into cells and the activity of firefly luciferase and Renilla luciferase was measured.

    Journal: Genome Research

    Article Title: Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

    doi: 10.1101/gr.230250.117

    Figure Lengend Snippet: CSDE1 is required for CVB3 infection. ( A ) Viability of wild-type (HeLa) and CSDE1 knockout (KO) cells after CVB3 infection. ( B ) Viral replication test of wild-type (HeLa) and CSDE1 KO cells using the CVB3 replicon. Transfection with cDNA encoding the eGFP-CSDE1 protein into CSDE1 KO cells rescued CVB3 replication. ( C ) Transfection with cDNA encoding the eGFP-CSDE1 protein into CSDE1 KO cells rescued CVB3 infection. After 8 h of CVB3 infection in wild-type (HeLa) and CSDE1 KO cells transfected with the plasmid encoding the eGFP-CSDE1 protein, cells were harvested and lysed for western blot analysis using anti-CSDE1 antibody, anti-VP1 antibody, and anti-beta actin antibody. ( D ) CVB3 IRES-dependent translation assay of wild-type (HeLa) and CSDE1 KO cells. Dual luciferase reporter plasmids containing the IRES sequence from five different viruses were transfected into cells and the activity of firefly luciferase and Renilla luciferase was measured.

    Article Snippet: A CSDE1-expressing plasmid was purchased form Vigene Bioscience ( ) and a plasmid encoding eGFP-CSDE1 was constructed using the eGFP-N1 plasmid (Clontech Laboratories). pCI-FLAG–ACBD3, which contains a FLAG-tagged ACBD3 gene, was obtained from Jun Sasaki (Fujita Health University, Aichi, Japan) ( ).

    Techniques: Infection, Knock-Out, Transfection, Plasmid Preparation, Western Blot, Luciferase, Sequencing, Activity Assay

    One step mini-chromosome construction by CRISPR-PCS. ( a ) One step mini-chromosome construction using CRISPR-PCS. The chromosomal region between C12-P1 and C12-P2 of Chr. XII was targeted to form a mini-chromosome. A 400 bp homology sequence was used in the splitting modules. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. Left panel, PFGE analysis of wild type FY834-Cas9 and 10 randomly chosen transformants. Right panel, Southern blot analysis after PFGE using probe 3 for detection of newly generated 31 kb mini-chromosome. The arrow beside the right panel represents the 31 kb expected mini-chromosome (lane 1, 3, 6, and 8). The 57 kb band in lane 2 and 7 show these strains have only one split at C12-P1. ( b ) One step mini-chromosome construction using CRISPR-PCS under the same experimental conditions as above except for use of a 50 bp homology sequence in the splitting module. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. The arrow beside the right panel represents the 31 kb expected mini-chromosome. The expected mini-chromosome was constructed in two strains (Lane 2 and 8). Four strains are shown to have single splitting at C12-P1 (Lane 3, 4, 6, and 9). Four strains are shown to have single splitting at C12-P2 (Lane 1, 5, 7, and 10). W. Wild type.

    Journal: Scientific Reports

    Article Title: CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae

    doi: 10.1038/srep30278

    Figure Lengend Snippet: One step mini-chromosome construction by CRISPR-PCS. ( a ) One step mini-chromosome construction using CRISPR-PCS. The chromosomal region between C12-P1 and C12-P2 of Chr. XII was targeted to form a mini-chromosome. A 400 bp homology sequence was used in the splitting modules. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. Left panel, PFGE analysis of wild type FY834-Cas9 and 10 randomly chosen transformants. Right panel, Southern blot analysis after PFGE using probe 3 for detection of newly generated 31 kb mini-chromosome. The arrow beside the right panel represents the 31 kb expected mini-chromosome (lane 1, 3, 6, and 8). The 57 kb band in lane 2 and 7 show these strains have only one split at C12-P1. ( b ) One step mini-chromosome construction using CRISPR-PCS under the same experimental conditions as above except for use of a 50 bp homology sequence in the splitting module. The splitting modules of C12-P1 and C12-P2 were marked with CgHIS3 and CgLEU2 , respectively. The arrow beside the right panel represents the 31 kb expected mini-chromosome. The expected mini-chromosome was constructed in two strains (Lane 2 and 8). Four strains are shown to have single splitting at C12-P1 (Lane 3, 4, 6, and 9). Four strains are shown to have single splitting at C12-P2 (Lane 1, 5, 7, and 10). W. Wild type.

    Article Snippet: CRISPR/Cas9 system in yeast The Streptococcus pyogenes Cas9 expressing plasmid (p414-TEF1p-Cas9-CYC1t) and gRNA expressing plasmid (p426-SNR52p-gRNA.CAN1.Y-SUP4t) were purchased from the AddGene repository ( http://www.addgene.org ).

    Techniques: CRISPR, Sequencing, Southern Blot, Generated, Construct

    Simultaneous double splitting by CRISPR-PCS. ( a ) Outline of CRISPR-PCS. One gRNA expressing plasmid for the specific targeting site and two splitting modules per target site are introduced into the FY834-Cas9 strain, which harbors a Cas9 expressing plasmid marked by TRP1 . The FY834-Cas9 strain was transformed with gRNA expressing plasmid with 20 bp specific target sequence and splitting modules. For splitting one genomic locus, one of the splitting modules contains either one of selective markers ( CgLEU2 , CgHIS3 , URA3 , and KanMX ) and the other module contains CEN4 as a centromere. Double strand breaks (DSBs) are induced in transformed cells by CRISPR/Cas9 near the targeted site followed by chromosome splitting by PCS. This combination of CRISPR/Cas9 and PCS is called CRISPR-PCS. After transformation, we selected by selective marker on splitting modules and Cas9 expressing plasmid. No selection of gRNA expressing plasmid was performed. Closed black circles represent the centromere. White boxes represent selective marker genes. Red and blue boxes represent the homology sequences for recombination. Arrows represent the telomere sequence. ( b ) Chromosome splitting by CRISPR-PCS. Position C16-P1 of Chr. XVI was chosen as the example. The number in parentheses represents the precise splitting point (the same shall apply hereinafter in all figures). The splitting module was marked with CgLEU2 gene. After splitting, two chromosomes (861 kb and 87 kb) are expected to be generated. Left panel, PFGE analysis of wild type FY834-Cas9 (WT) and 10 randomly chosen transformants (lanes 1–10). Right panel, Southern blot analysis after PFGE using probe 1 for detection of the newly generated 87 kb chromosome. ( c ) Simultaneous double splitting in different chromosomes. Position C16-P1 of Chr. XVI and position C15-P1 of Chr. XV were simultaneously split by CRISPR-PCS. The splitting modules of C15-P1 and C16-P1 were marked with CgHIS3 and CgLEU2 , respectively. After splitting, four derived chromosomes are expected: 861 kb and 87 kb derivatives from Chr. XVI and 208 kb and 883 kb derivatives from Chr. XV. Left panel, PFGE analysis of wild type FY834-Cas9 (WT) and 10 randomly chosen transformants. Middle panel and right panel, Southern blot analysis using probe 1 for detecting the newly generated 87 kb chromosome and probe 2 for detecting the newly generated 208 kb chromosome.

    Journal: Scientific Reports

    Article Title: CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae

    doi: 10.1038/srep30278

    Figure Lengend Snippet: Simultaneous double splitting by CRISPR-PCS. ( a ) Outline of CRISPR-PCS. One gRNA expressing plasmid for the specific targeting site and two splitting modules per target site are introduced into the FY834-Cas9 strain, which harbors a Cas9 expressing plasmid marked by TRP1 . The FY834-Cas9 strain was transformed with gRNA expressing plasmid with 20 bp specific target sequence and splitting modules. For splitting one genomic locus, one of the splitting modules contains either one of selective markers ( CgLEU2 , CgHIS3 , URA3 , and KanMX ) and the other module contains CEN4 as a centromere. Double strand breaks (DSBs) are induced in transformed cells by CRISPR/Cas9 near the targeted site followed by chromosome splitting by PCS. This combination of CRISPR/Cas9 and PCS is called CRISPR-PCS. After transformation, we selected by selective marker on splitting modules and Cas9 expressing plasmid. No selection of gRNA expressing plasmid was performed. Closed black circles represent the centromere. White boxes represent selective marker genes. Red and blue boxes represent the homology sequences for recombination. Arrows represent the telomere sequence. ( b ) Chromosome splitting by CRISPR-PCS. Position C16-P1 of Chr. XVI was chosen as the example. The number in parentheses represents the precise splitting point (the same shall apply hereinafter in all figures). The splitting module was marked with CgLEU2 gene. After splitting, two chromosomes (861 kb and 87 kb) are expected to be generated. Left panel, PFGE analysis of wild type FY834-Cas9 (WT) and 10 randomly chosen transformants (lanes 1–10). Right panel, Southern blot analysis after PFGE using probe 1 for detection of the newly generated 87 kb chromosome. ( c ) Simultaneous double splitting in different chromosomes. Position C16-P1 of Chr. XVI and position C15-P1 of Chr. XV were simultaneously split by CRISPR-PCS. The splitting modules of C15-P1 and C16-P1 were marked with CgHIS3 and CgLEU2 , respectively. After splitting, four derived chromosomes are expected: 861 kb and 87 kb derivatives from Chr. XVI and 208 kb and 883 kb derivatives from Chr. XV. Left panel, PFGE analysis of wild type FY834-Cas9 (WT) and 10 randomly chosen transformants. Middle panel and right panel, Southern blot analysis using probe 1 for detecting the newly generated 87 kb chromosome and probe 2 for detecting the newly generated 208 kb chromosome.

    Article Snippet: CRISPR/Cas9 system in yeast The Streptococcus pyogenes Cas9 expressing plasmid (p414-TEF1p-Cas9-CYC1t) and gRNA expressing plasmid (p426-SNR52p-gRNA.CAN1.Y-SUP4t) were purchased from the AddGene repository ( http://www.addgene.org ).

    Techniques: CRISPR, Expressing, Plasmid Preparation, Transformation Assay, Sequencing, Marker, Selection, Generated, Southern Blot, Derivative Assay

    Simultaneous multiple splitting. ( a ) Simultaneous triple splitting by CRISPR-PCS. Three sites (C16-P1, C15-P1, and C4-P3) were targeted. A 50 bp homology sequence was used in the splitting module. The splitting modules of C16-P1, C15-P1, and C4-P3 were marked with CgLEU2, CgHIS3, and URA3 , respectively. Wild type FY834-Cas9 and four randomly chosen transformants were subjected to PFGE and subsequent Southern blot analysis. Probes 1, 2, and 4 were used to detect Chr. XVI, Chr. XV, and Chr. IV respectively. ( b ) One step construction of four chromosomes from one chromosome. Chr. IV was targeted to split at three positions (C4-P1, C4-P2, and C4-P3). A 50 bp homology sequence was used in the splitting module. The splitting modules of C4-P1, C4-P2, and C4-P3 were marked with CgHIS3, CgLEU2, and URA3 , respectively. Wild type FY834-Cas9 and two randomly chosen transformants were subjected to PFGE and subsequent Southern blot analysis. Probes 5, 6, 7, and 4 were used to detect the 522 kb, 79 kb, 398 kb, and 533 kb chromosomes, respectively. ( c ) Simultaneous quadruple splitting by CRISPR-PCS. Four sites (C16-P1, C15-P1, C12-P1, and C4-P3) were targeted. A 50 kb homology sequence was used in the splitting module. The splitting modules of C16-P1, C15-P1, C12-P1, and C4-P3 were marked with CgLEU2, CgHIS3, KanMX , and URA3 , respectively. Wild type FY834-Cas9 and four randomly chosen transformants were subjected to PFGE and subsequent Southern blot analysis. Probes 1, 2, 3, and 4 were used to detect Chr. XVI, XV, XII, and IV, respectively.

    Journal: Scientific Reports

    Article Title: CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae

    doi: 10.1038/srep30278

    Figure Lengend Snippet: Simultaneous multiple splitting. ( a ) Simultaneous triple splitting by CRISPR-PCS. Three sites (C16-P1, C15-P1, and C4-P3) were targeted. A 50 bp homology sequence was used in the splitting module. The splitting modules of C16-P1, C15-P1, and C4-P3 were marked with CgLEU2, CgHIS3, and URA3 , respectively. Wild type FY834-Cas9 and four randomly chosen transformants were subjected to PFGE and subsequent Southern blot analysis. Probes 1, 2, and 4 were used to detect Chr. XVI, Chr. XV, and Chr. IV respectively. ( b ) One step construction of four chromosomes from one chromosome. Chr. IV was targeted to split at three positions (C4-P1, C4-P2, and C4-P3). A 50 bp homology sequence was used in the splitting module. The splitting modules of C4-P1, C4-P2, and C4-P3 were marked with CgHIS3, CgLEU2, and URA3 , respectively. Wild type FY834-Cas9 and two randomly chosen transformants were subjected to PFGE and subsequent Southern blot analysis. Probes 5, 6, 7, and 4 were used to detect the 522 kb, 79 kb, 398 kb, and 533 kb chromosomes, respectively. ( c ) Simultaneous quadruple splitting by CRISPR-PCS. Four sites (C16-P1, C15-P1, C12-P1, and C4-P3) were targeted. A 50 kb homology sequence was used in the splitting module. The splitting modules of C16-P1, C15-P1, C12-P1, and C4-P3 were marked with CgLEU2, CgHIS3, KanMX , and URA3 , respectively. Wild type FY834-Cas9 and four randomly chosen transformants were subjected to PFGE and subsequent Southern blot analysis. Probes 1, 2, 3, and 4 were used to detect Chr. XVI, XV, XII, and IV, respectively.

    Article Snippet: CRISPR/Cas9 system in yeast The Streptococcus pyogenes Cas9 expressing plasmid (p414-TEF1p-Cas9-CYC1t) and gRNA expressing plasmid (p426-SNR52p-gRNA.CAN1.Y-SUP4t) were purchased from the AddGene repository ( http://www.addgene.org ).

    Techniques: CRISPR, Sequencing, Southern Blot

    ex14D-EZH2 participates in establishing and maintaining H3K27 methylation and promotes ES cell differentiation. a A schematic of how ex14D- Ezh2 ES cell lines were created by CRISPR-Cas9. b Western blot analysis of EZH2 proteins in ex14D- Ezh2 cells. c Western blot analysis of histone H3K27 methylation levels in ex14D- Ezh2 ES cells. d A smear plot showing ex14D-EZH2 is not sufficient for saturating EZH2 targeting genes. e A Venn diagram showing that ex14D-EZH2 binds to the majority EZH2 targets. f Assessment of H3K27me3 maintenance on key developmental genes in ex14D- Ezh2 ES cells by ChIP-PCR analysis. g Assessment of the newly introduced H3K27me3 marks during ES cell differentiation. h qPCR analysis of the expression of mesoderm marker genes during LIF withdrawal-induced ES cell differentiation

    Journal: Epigenetics & Chromatin

    Article Title: EZH2 variants differentially regulate polycomb repressive complex 2 in histone methylation and cell differentiation

    doi: 10.1186/s13072-018-0242-9

    Figure Lengend Snippet: ex14D-EZH2 participates in establishing and maintaining H3K27 methylation and promotes ES cell differentiation. a A schematic of how ex14D- Ezh2 ES cell lines were created by CRISPR-Cas9. b Western blot analysis of EZH2 proteins in ex14D- Ezh2 cells. c Western blot analysis of histone H3K27 methylation levels in ex14D- Ezh2 ES cells. d A smear plot showing ex14D-EZH2 is not sufficient for saturating EZH2 targeting genes. e A Venn diagram showing that ex14D-EZH2 binds to the majority EZH2 targets. f Assessment of H3K27me3 maintenance on key developmental genes in ex14D- Ezh2 ES cells by ChIP-PCR analysis. g Assessment of the newly introduced H3K27me3 marks during ES cell differentiation. h qPCR analysis of the expression of mesoderm marker genes during LIF withdrawal-induced ES cell differentiation

    Article Snippet: Briefly, 5 × 104 E14 ES cells were cultured on 60 mm dishes for 1 day and then transfected with plasmids expressing Cas9 and sgRNAs, along with a plasmid expressing PGK-PuroR (Addgene, Cat. No. 31937) using FuGENE HD reagent (Promega) according to the manufacturer’s instructions.

    Techniques: Methylation, Cell Differentiation, CRISPR, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Marker

    1,25(OH) 2 D 2 inhibits LPS-induced COX-2 and phospho-AKT T308 in the RAW264. 7 cells. RAW264.7 cells were exposed to 250 n m 1,25(OH) 2 D 2 or VDR siRNAs ( si-VDR ), and cell lysates were analyzed to Western blotting. The experiments were repeated three times.

    Journal: The Journal of Biological Chemistry

    Article Title: Vitamin D Inhibits COX-2 Expression and Inflammatory Response by Targeting Thioesterase Superfamily Member 4 *

    doi: 10.1074/jbc.M113.517581

    Figure Lengend Snippet: 1,25(OH) 2 D 2 inhibits LPS-induced COX-2 and phospho-AKT T308 in the RAW264. 7 cells. RAW264.7 cells were exposed to 250 n m 1,25(OH) 2 D 2 or VDR siRNAs ( si-VDR ), and cell lysates were analyzed to Western blotting. The experiments were repeated three times.

    Article Snippet: RAW264.7 and HEK293-A cells were transfected with VDR expression plasmids (GeneCopoeia Inc., Rockville, MD) using the LipofectamineTM 2000 reagent (Invitrogen).

    Techniques: Western Blot

    Effects of GNL3 knockdown and overexpression on the levels of the EMT-related markers, β-catenin and GNL3 in vitro . (A and B) In HCT-116 cells, GNL3 knockdown (GNL3 shRNA) increased E-cadherin levels and reduced N-cadherin, vimentin, nuclear β-catenin (β-catenin-N) and GNL3 levels compared with controls (Con). (A and C) In HT-29 cells, GNL3 overexpression (GNL3) increased the N-cadherin, vimentin, β-catenin-N and GNL3 levels and reduced the E-cadherin levels compared with the negative controls (NC). **P

    Journal: Oncology Reports

    Article Title: Upregulation of GNL3 expression promotes colon cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

    doi: 10.3892/or.2017.5923

    Figure Lengend Snippet: Effects of GNL3 knockdown and overexpression on the levels of the EMT-related markers, β-catenin and GNL3 in vitro . (A and B) In HCT-116 cells, GNL3 knockdown (GNL3 shRNA) increased E-cadherin levels and reduced N-cadherin, vimentin, nuclear β-catenin (β-catenin-N) and GNL3 levels compared with controls (Con). (A and C) In HT-29 cells, GNL3 overexpression (GNL3) increased the N-cadherin, vimentin, β-catenin-N and GNL3 levels and reduced the E-cadherin levels compared with the negative controls (NC). **P

    Article Snippet: GNL3 knockdown plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA; TRCN0000293740) and GNL3 expression plasmids were purchased from Sino Biological Inc. (Beijing, China; HG12415-UT).

    Techniques: Over Expression, In Vitro, shRNA

    Treatment with suitable concentrations of LiCl activates the Wnt/β-catenin signaling pathway, and LiCl partially reverses the GNL3 knockdown-induced inhibition of the EMT and Wnt/β-catenin signaling pathway in HCT-116 cells. (A and B) Both 20 and 40 mM LiCl were appropriate concentrations to activate the Wnt/β-catenin signaling pathway. (C and D) GNL3 knockdown (GNL3 shRNA) increased E-cadherin expression and decreased N-cadherin, vimentin, nuclear β-catenin (β-catenin-N) and GNL3 expression compared with the controls (Con). LiCl increased N-cadherin, vimentin and β-catenin-N expression and reduced E-cadherin expression compared with the GNL3 shRNA group. *P

    Journal: Oncology Reports

    Article Title: Upregulation of GNL3 expression promotes colon cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

    doi: 10.3892/or.2017.5923

    Figure Lengend Snippet: Treatment with suitable concentrations of LiCl activates the Wnt/β-catenin signaling pathway, and LiCl partially reverses the GNL3 knockdown-induced inhibition of the EMT and Wnt/β-catenin signaling pathway in HCT-116 cells. (A and B) Both 20 and 40 mM LiCl were appropriate concentrations to activate the Wnt/β-catenin signaling pathway. (C and D) GNL3 knockdown (GNL3 shRNA) increased E-cadherin expression and decreased N-cadherin, vimentin, nuclear β-catenin (β-catenin-N) and GNL3 expression compared with the controls (Con). LiCl increased N-cadherin, vimentin and β-catenin-N expression and reduced E-cadherin expression compared with the GNL3 shRNA group. *P

    Article Snippet: GNL3 knockdown plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA; TRCN0000293740) and GNL3 expression plasmids were purchased from Sino Biological Inc. (Beijing, China; HG12415-UT).

    Techniques: Inhibition, shRNA, Expressing

    Immunofluorescence staining for β-catenin. The β-catenin proteins labeled in red and the nuclei were stained with DAPI and are labeled in blue. GNL3 knockdown (GNL3 shRNA) in HCT-116 cells reduced the nuclear β-catenin levels compared with the controls (Con). However, GNL3 overexpression (GNL3) in HT-29 cells increased the nuclear β-catenin levels compared with the negative controls (NC). Original magnification, ×400.

    Journal: Oncology Reports

    Article Title: Upregulation of GNL3 expression promotes colon cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

    doi: 10.3892/or.2017.5923

    Figure Lengend Snippet: Immunofluorescence staining for β-catenin. The β-catenin proteins labeled in red and the nuclei were stained with DAPI and are labeled in blue. GNL3 knockdown (GNL3 shRNA) in HCT-116 cells reduced the nuclear β-catenin levels compared with the controls (Con). However, GNL3 overexpression (GNL3) in HT-29 cells increased the nuclear β-catenin levels compared with the negative controls (NC). Original magnification, ×400.

    Article Snippet: GNL3 knockdown plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA; TRCN0000293740) and GNL3 expression plasmids were purchased from Sino Biological Inc. (Beijing, China; HG12415-UT).

    Techniques: Immunofluorescence, Staining, Labeling, shRNA, Over Expression

    Effects of GNL3 knockdown and overexpression on cell invasion and migration in vitro . (A) Following GNL3 knockdown (GNL3 shRNA), the invasive capacity of HCT-116 cells was decreased compared with those of control cells (Con). Following GNL3 overexpression (GNL3), the invasive capacity of HT-29 cells was increased compared with negative control cells (NC). (B) GNL3 knockdown inhibited the migration of HCT-116 cells, and GNL3 overexpression promoted the migration of HT-29 cells. *P

    Journal: Oncology Reports

    Article Title: Upregulation of GNL3 expression promotes colon cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

    doi: 10.3892/or.2017.5923

    Figure Lengend Snippet: Effects of GNL3 knockdown and overexpression on cell invasion and migration in vitro . (A) Following GNL3 knockdown (GNL3 shRNA), the invasive capacity of HCT-116 cells was decreased compared with those of control cells (Con). Following GNL3 overexpression (GNL3), the invasive capacity of HT-29 cells was increased compared with negative control cells (NC). (B) GNL3 knockdown inhibited the migration of HCT-116 cells, and GNL3 overexpression promoted the migration of HT-29 cells. *P

    Article Snippet: GNL3 knockdown plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA; TRCN0000293740) and GNL3 expression plasmids were purchased from Sino Biological Inc. (Beijing, China; HG12415-UT).

    Techniques: Over Expression, Migration, In Vitro, shRNA, Negative Control

    GNL3 expression and its clinical significance in colon cancer. (A) Representative images of immunohistochemical staining for GNL3 in normal, tumor-adjacent tissues and colon cancer tissues. Original magnification, ×200. (B) GNL3 levels in normal, tumor-adjacent tissues (N1-4) and colon cancer tissues (T1-4) were detected by western blotting. According to the quantitative analysis, GNL3 was expressed at significantly higher levels in colon cancer tissues than in normal, tumor-adjacent tissues. (C and D) Overall 5-year and disease-free survival curves for GNL3-negative and GNL3-positive patients with colon cancer according to the Kaplan-Meier analysis. The overall and disease-free survival rates of the GNL3-positive groups were markedly reduced compared with the GNL3-negative groups. **P

    Journal: Oncology Reports

    Article Title: Upregulation of GNL3 expression promotes colon cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

    doi: 10.3892/or.2017.5923

    Figure Lengend Snippet: GNL3 expression and its clinical significance in colon cancer. (A) Representative images of immunohistochemical staining for GNL3 in normal, tumor-adjacent tissues and colon cancer tissues. Original magnification, ×200. (B) GNL3 levels in normal, tumor-adjacent tissues (N1-4) and colon cancer tissues (T1-4) were detected by western blotting. According to the quantitative analysis, GNL3 was expressed at significantly higher levels in colon cancer tissues than in normal, tumor-adjacent tissues. (C and D) Overall 5-year and disease-free survival curves for GNL3-negative and GNL3-positive patients with colon cancer according to the Kaplan-Meier analysis. The overall and disease-free survival rates of the GNL3-positive groups were markedly reduced compared with the GNL3-negative groups. **P

    Article Snippet: GNL3 knockdown plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA; TRCN0000293740) and GNL3 expression plasmids were purchased from Sino Biological Inc. (Beijing, China; HG12415-UT).

    Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

    GNL3 expression was assessed in different colon cancer cell lines using western blotting. (A) GNL3 levels in five colon cancer cell lines: HCT-116, HT-29, SW620, Caco-2 and LoVo cells. (B) GNL3 expression was significantly reduced in GNL3 knockdown cells (GNL3 shRNA) compared with that in the control cells (Con) and was substantially increased in GNL3-overexpressing cells (GNL3) compared with that in the negative control cells (NC). **P

    Journal: Oncology Reports

    Article Title: Upregulation of GNL3 expression promotes colon cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

    doi: 10.3892/or.2017.5923

    Figure Lengend Snippet: GNL3 expression was assessed in different colon cancer cell lines using western blotting. (A) GNL3 levels in five colon cancer cell lines: HCT-116, HT-29, SW620, Caco-2 and LoVo cells. (B) GNL3 expression was significantly reduced in GNL3 knockdown cells (GNL3 shRNA) compared with that in the control cells (Con) and was substantially increased in GNL3-overexpressing cells (GNL3) compared with that in the negative control cells (NC). **P

    Article Snippet: GNL3 knockdown plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA; TRCN0000293740) and GNL3 expression plasmids were purchased from Sino Biological Inc. (Beijing, China; HG12415-UT).

    Techniques: Expressing, Western Blot, shRNA, Negative Control

    Effects of GNL3 knockdown and overexpression on cell proliferation and colony formation in vitro and tumor growth in nude mice in vivo . (A) In HCT-116 cells, the proliferation rates of the GNL3 knockdown group (GNL3 shRNA) were lower than the control group (Con). (B) In HT-29 cells, the proliferation rates of the GNL3-overexpressing group (GNL3) were much higher than in the negative control group (NC). (C) GNL3 knockdown inhibited the formation of HCT-116 cell colonies, and GNL3 overexpression promoted the formation of HT-29 cell colonies. (D and E) Representative xenograft tumors are shown and the tumor volume was measured weekly. *P

    Journal: Oncology Reports

    Article Title: Upregulation of GNL3 expression promotes colon cancer cell proliferation, migration, invasion and epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

    doi: 10.3892/or.2017.5923

    Figure Lengend Snippet: Effects of GNL3 knockdown and overexpression on cell proliferation and colony formation in vitro and tumor growth in nude mice in vivo . (A) In HCT-116 cells, the proliferation rates of the GNL3 knockdown group (GNL3 shRNA) were lower than the control group (Con). (B) In HT-29 cells, the proliferation rates of the GNL3-overexpressing group (GNL3) were much higher than in the negative control group (NC). (C) GNL3 knockdown inhibited the formation of HCT-116 cell colonies, and GNL3 overexpression promoted the formation of HT-29 cell colonies. (D and E) Representative xenograft tumors are shown and the tumor volume was measured weekly. *P

    Article Snippet: GNL3 knockdown plasmids were purchased from Sigma-Aldrich (St. Louis, MO, USA; TRCN0000293740) and GNL3 expression plasmids were purchased from Sino Biological Inc. (Beijing, China; HG12415-UT).

    Techniques: Over Expression, In Vitro, Mouse Assay, In Vivo, shRNA, Negative Control

    HDAC4 is a direct target of miR-365. A ) Diagram of predicted miR-365 sequential pairing with a putative target region (miR-365 binding site) in the 3′-UTR of HDAC4 mRNA. The 5′ end of miR-365 contains a base pair sequence complementary

    Journal: The FASEB Journal

    Article Title: MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4

    doi: 10.1096/fj.11-185132

    Figure Lengend Snippet: HDAC4 is a direct target of miR-365. A ) Diagram of predicted miR-365 sequential pairing with a putative target region (miR-365 binding site) in the 3′-UTR of HDAC4 mRNA. The 5′ end of miR-365 contains a base pair sequence complementary

    Article Snippet: HDAC4 expression plasmids were purchased from ADDgene (Cambridge, MA, USA). pGL3-RunX2-Luc (0.6 kb of Rat Runx2 promoter) was kindly provided by Dr. Gary Stein (University of Massachusetts Medical School, Worcester, MA, USA; ref. ). pGL-HDAC4 was kindly provided by Dr. Da-Zhi Wang (Children's Hospital Boston and Harvard Medical School, Boston, MA, USA; ref. ).

    Techniques: Binding Assay, Sequencing

    Overexpression of HDAC4 inhibits miR-365 stimulation of Ihh and Col X mRNA levels. A ) Primary mouse chondrocytes were transfected with following 3 groups of constructs. Control: a control miRNA (conmiR) and a cDNA plasmid containing empty vector (EV);

    Journal: The FASEB Journal

    Article Title: MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4

    doi: 10.1096/fj.11-185132

    Figure Lengend Snippet: Overexpression of HDAC4 inhibits miR-365 stimulation of Ihh and Col X mRNA levels. A ) Primary mouse chondrocytes were transfected with following 3 groups of constructs. Control: a control miRNA (conmiR) and a cDNA plasmid containing empty vector (EV);

    Article Snippet: HDAC4 expression plasmids were purchased from ADDgene (Cambridge, MA, USA). pGL3-RunX2-Luc (0.6 kb of Rat Runx2 promoter) was kindly provided by Dr. Gary Stein (University of Massachusetts Medical School, Worcester, MA, USA; ref. ). pGL-HDAC4 was kindly provided by Dr. Da-Zhi Wang (Children's Hospital Boston and Harvard Medical School, Boston, MA, USA; ref. ).

    Techniques: Over Expression, Transfection, Construct, Plasmid Preparation

    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). pGL3 + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).

    Journal: PLoS Genetics

    Article Title: New Small Nuclear RNA Gene-Like Transcriptional Units as Sources of Regulatory Transcripts

    doi: 10.1371/journal.pgen.0030001

    Figure Lengend Snippet: Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). pGL3 + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).

    Article Snippet: Oligos used to subclone the novel pol III type III promoters within Not I/HinD III restriction sites (uppercase letters) were the following: 11AFprom Not I: 5′-atgcGCGGCCGCatttgcatgtcgctatgtg-3′ 11ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccacgcactcagctcgtg-3′ 14AFprom Not I: 5′-atgcGCGGCCGCaactgatgtatgattatatctt-3′ 14ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccattattatctcctttgttctgt-3′ 21AFprom Not I: 5′-atgcGCGGCCGCacagctgtagcagatgct-3′ 21ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccaccacacttggtcaactat-3′ 29AFprom Not I: 5′-atgcGCGGCCGCttctcacctaaaggagtc-3′ 29ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccttctaatcctcctaagatca-3′ 38AFprom Not I: 5′-atgcGCGGCCGCttcactaagatccagtgc-3′ 38Arprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccgattcatgaacacagaatatt3′ 51AFprom Not I: 5′-atgcGCGGCCGCgttgaacatttaactctgtat-3′ 51Arprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccctcatggcacttggagat-3′ In this analysis, the above constructs were cotransfected with a pGL3 plasmid-expressing (Promega) firefly luciferase as a target to be silenced and with a pRL plasmid expressing (Promega) renilla luciferase to which all the determinations were normalized.

    Techniques: Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Activity Assay, Transfection, Luciferase, Construct, Sequencing, Negative Control, Quantitative RT-PCR

    HEK293T cells were transiently transfected with 80 ng of p(Gal) 2 -50.huIL6P-luc+ and with various expression plasmids, the total amount of DNA being fixed at 400 ng, i.e., pGal4 (40 ng) or pGal4-p65 (10 ng) ( A ) or pGal4-VP16 (40 ng) ( B ), whether or not with pCMV-CBP (25 ng or 100 ng) and/or pSVhGRα (25 ng or 100 ng). In control lanes with CBP and GR, + corresponds to the highest concentration. Assays were performed in triplicate and are representative of two independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Glucocorticoids repress NF-?B-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell

    doi:

    Figure Lengend Snippet: HEK293T cells were transiently transfected with 80 ng of p(Gal) 2 -50.huIL6P-luc+ and with various expression plasmids, the total amount of DNA being fixed at 400 ng, i.e., pGal4 (40 ng) or pGal4-p65 (10 ng) ( A ) or pGal4-VP16 (40 ng) ( B ), whether or not with pCMV-CBP (25 ng or 100 ng) and/or pSVhGRα (25 ng or 100 ng). In control lanes with CBP and GR, + corresponds to the highest concentration. Assays were performed in triplicate and are representative of two independent experiments.

    Article Snippet: The ICAM promoter, kindly provided by K. Roebuck (Rush Presbyterian St. Luke's Medical Center, Chicago) was used to construct pICAM-luc as described ( ). pCMV-CBP, pRSV and pRSVp65, pSVhGRα, and PCR3.1-SRC1a were kind gifts from R. Eckner (Institute for Molecular Biology, Zurich), G. Manfioletti (University of Trieste, Italy), W. Rombauts (University of Leuven, Belgium), and B. W. O'Malley (Baylor College of Medicine, Houston), respectively. pcDNA3, used as an empty control vector for the CBP-expressing plasmid, was purchased from Invitrogen. pGal4, pGal4-p65, and pGal4-VP16 were generously provided by M. L. Schmitz (German Cancer Research Center, Heidelberg) and, together with p(Gal)2 -50hu.IL6P-luc+, have been described ( , ).

    Techniques: Transfection, Expressing, Concentration Assay

    ( A ) Immunoprecipitation with anti-p65 (lanes 1–4 and 6–8) or an irrelevant antibody (lane 5) of cell lysates, transfected with pRSV-p65 (200 ng), pCMV-CBP (1 μg), and/or pSVhGRα (200 ng or 400 ng), followed by Western blot analysis with anti-CBP antibody. Lane 4 is a control with lysis buffer only. The input lane represents one-third of the amount used in the assay. The M lane represents molecular mass markers. ( B ) Similar lysate preparation to which increasing amounts (3, 5, and 10 μl corresponding to approximately 30, 50, and 100 ng) of in vitro translated GR protein (GR*) are added, followed by immunoprecipitation and Western blot analysis as described for A . The upper arrow marks the 265-kDa band corresponding with coimmunoprecipitated CBP protein. NS, nonspecific band. The result is representative of three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Glucocorticoids repress NF-?B-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell

    doi:

    Figure Lengend Snippet: ( A ) Immunoprecipitation with anti-p65 (lanes 1–4 and 6–8) or an irrelevant antibody (lane 5) of cell lysates, transfected with pRSV-p65 (200 ng), pCMV-CBP (1 μg), and/or pSVhGRα (200 ng or 400 ng), followed by Western blot analysis with anti-CBP antibody. Lane 4 is a control with lysis buffer only. The input lane represents one-third of the amount used in the assay. The M lane represents molecular mass markers. ( B ) Similar lysate preparation to which increasing amounts (3, 5, and 10 μl corresponding to approximately 30, 50, and 100 ng) of in vitro translated GR protein (GR*) are added, followed by immunoprecipitation and Western blot analysis as described for A . The upper arrow marks the 265-kDa band corresponding with coimmunoprecipitated CBP protein. NS, nonspecific band. The result is representative of three independent experiments.

    Article Snippet: The ICAM promoter, kindly provided by K. Roebuck (Rush Presbyterian St. Luke's Medical Center, Chicago) was used to construct pICAM-luc as described ( ). pCMV-CBP, pRSV and pRSVp65, pSVhGRα, and PCR3.1-SRC1a were kind gifts from R. Eckner (Institute for Molecular Biology, Zurich), G. Manfioletti (University of Trieste, Italy), W. Rombauts (University of Leuven, Belgium), and B. W. O'Malley (Baylor College of Medicine, Houston), respectively. pcDNA3, used as an empty control vector for the CBP-expressing plasmid, was purchased from Invitrogen. pGal4, pGal4-p65, and pGal4-VP16 were generously provided by M. L. Schmitz (German Cancer Research Center, Heidelberg) and, together with p(Gal)2 -50hu.IL6P-luc+, have been described ( , ).

    Techniques: Immunoprecipitation, Transfection, Western Blot, Lysis, In Vitro

    ( A ) HEK293T cells were transiently transfected with 80 ng of p1168hu.IL6P-luc+ and with pRSV-p65 (20 ng), pSVhGRα (100 to 200 ng), pCMV-CBP (as indicated), and/or pcDNA3 or pRSV, keeping the total amount of DNA constant at 600 ng per 24-well plate. Cell lysates were assayed for luc activities and normalized for protein content. Promoter activities are expressed as “induction factor,” i.e., the ratio of expression levels recorded either under induced and noninduced conditions or under transfected and mock-transfected conditions. Assays were performed in triplicate, and results are representative of at least four independent transfection experiments. ( B ) Western blot analysis of lysates of transfected cells corresponding to 25 μg of protein content (the luc activities had a similar regulation on a full IL-6 promoter-dependent reporter gene as shown in A ). Samples were transfected with 100 ng (lane 4) or 200 ng (lanes 5–7) of pCMV-CBP or 25 ng (lane 6) or 100 ng (lanes 3 and 7) pSVhGRα. The membrane was probed with an anti-p65 rabbit polyclonal antibody. ( C ) Samples were transfected as described for B (except for lane 2 where a setup with 50 ng of pCMV-CBP was included), blotted, and probed with an anti-CBP rabbit polyclonal antibody.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Glucocorticoids repress NF-?B-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell

    doi:

    Figure Lengend Snippet: ( A ) HEK293T cells were transiently transfected with 80 ng of p1168hu.IL6P-luc+ and with pRSV-p65 (20 ng), pSVhGRα (100 to 200 ng), pCMV-CBP (as indicated), and/or pcDNA3 or pRSV, keeping the total amount of DNA constant at 600 ng per 24-well plate. Cell lysates were assayed for luc activities and normalized for protein content. Promoter activities are expressed as “induction factor,” i.e., the ratio of expression levels recorded either under induced and noninduced conditions or under transfected and mock-transfected conditions. Assays were performed in triplicate, and results are representative of at least four independent transfection experiments. ( B ) Western blot analysis of lysates of transfected cells corresponding to 25 μg of protein content (the luc activities had a similar regulation on a full IL-6 promoter-dependent reporter gene as shown in A ). Samples were transfected with 100 ng (lane 4) or 200 ng (lanes 5–7) of pCMV-CBP or 25 ng (lane 6) or 100 ng (lanes 3 and 7) pSVhGRα. The membrane was probed with an anti-p65 rabbit polyclonal antibody. ( C ) Samples were transfected as described for B (except for lane 2 where a setup with 50 ng of pCMV-CBP was included), blotted, and probed with an anti-CBP rabbit polyclonal antibody.

    Article Snippet: The ICAM promoter, kindly provided by K. Roebuck (Rush Presbyterian St. Luke's Medical Center, Chicago) was used to construct pICAM-luc as described ( ). pCMV-CBP, pRSV and pRSVp65, pSVhGRα, and PCR3.1-SRC1a were kind gifts from R. Eckner (Institute for Molecular Biology, Zurich), G. Manfioletti (University of Trieste, Italy), W. Rombauts (University of Leuven, Belgium), and B. W. O'Malley (Baylor College of Medicine, Houston), respectively. pcDNA3, used as an empty control vector for the CBP-expressing plasmid, was purchased from Invitrogen. pGal4, pGal4-p65, and pGal4-VP16 were generously provided by M. L. Schmitz (German Cancer Research Center, Heidelberg) and, together with p(Gal)2 -50hu.IL6P-luc+, have been described ( , ).

    Techniques: Transfection, Expressing, Western Blot