β globin genes Search Results


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  • 88
    Stratagene rabbit β globin gene
    Association of TTP with ARE-containing mRNAs. ( A ) The TNFα ARE-containing <t>β-globin</t> reporter (TNF-UTR) or the control reporter (TNF-ΔARE), as shown in the top schematics, was transfected into HeLa cells. The cell lysates were subjected to immunoprecipitation using anti-TRN. Precipitated RNAs were analyzed by RT–PCR using primers specific to the reporters. ( B ) HeLa cells were mock-transfected or transiently transfected with siRNA against TRN. Depletion of TRN was examined by immunoblotting using anti-TRN. For mRNA stability analysis, the reporters used were the same as those in A, and meanwhile a GFP expression vector was cotransfected as a reference. Cells were harvested at the indicated time points after doxycycline addition. RNase protection was performed using 32 P-labeled probes specific to the reporter or the GFP mRNA. The graph shows the level of reporter mRNA remaining at each time point relative to that of the GFP reference. Arbitrary mRNA intensity was determined by phosphoimaging. Average and standard deviation were obtained from three independent experiments. The representative gel shows an experiment using the TNF-UTR reporter.
    Rabbit β Globin Gene, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche β globin gene
    Association of TTP with ARE-containing mRNAs. ( A ) The TNFα ARE-containing <t>β-globin</t> reporter (TNF-UTR) or the control reporter (TNF-ΔARE), as shown in the top schematics, was transfected into HeLa cells. The cell lysates were subjected to immunoprecipitation using anti-TRN. Precipitated RNAs were analyzed by RT–PCR using primers specific to the reporters. ( B ) HeLa cells were mock-transfected or transiently transfected with siRNA against TRN. Depletion of TRN was examined by immunoblotting using anti-TRN. For mRNA stability analysis, the reporters used were the same as those in A, and meanwhile a GFP expression vector was cotransfected as a reference. Cells were harvested at the indicated time points after doxycycline addition. RNase protection was performed using 32 P-labeled probes specific to the reporter or the GFP mRNA. The graph shows the level of reporter mRNA remaining at each time point relative to that of the GFP reference. Arbitrary mRNA intensity was determined by phosphoimaging. Average and standard deviation were obtained from three independent experiments. The representative gel shows an experiment using the TNF-UTR reporter.
    β Globin Gene, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega β globin genes
    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and <t>β-globin</t> primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
    β Globin Genes, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β globin gene
    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and <t>β-globin</t> primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
    β Globin Gene, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Glaxo Smith β globin gene
    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and <t>β-globin</t> primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
    β Globin Gene, supplied by Glaxo Smith, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Metabion International AG β globin gene
    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and <t>β-globin</t> primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
    β Globin Gene, supplied by Metabion International AG, used in various techniques. Bioz Stars score: 93/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher β globin gene
    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and <t>β-globin</t> primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.
    β Globin Gene, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene β globin gene
    Recognition of the MTRR pseudoexon with weak 5′ splice site is dependent on the presence of the c.903+469T > C mutation. A: Splicing minigene assay. Upper panel depicts the <t>β-globin</t> and HIV- tat minigenes harboring the wild-type or mutant MTRR pseudoexon. Minigenes were used to transiently transfect COS-7 cells. After RNA isolation the splicing products were analyzed by RT-PCR using minigene-specific primers. The lower bands represent correctly spliced minigene exons, the upper bands represent MTRR pseudoexon inserted between minigene exons. B: 5′ splice site optimization. The suboptimal pseudoexon 5′ splice site ( A AG/gt c ag c ) was converted to an optimal 5′ splice site (variant +3A: C AG/gt a ag t ) or to the nearly optimal 5′ splice site (variant 13G: C AG/gt g ag t ), and wild-type and mutant minigenes were analyzed by transfection/RT-PCR. The scores of the different 5′ splice sites assessed by the MaxEntScan program ( http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html ) [ Yeo and Burge, 2004 ] are shown in the left panel. Right panel shows results from RT-PCR analysis of splicing products.
    β Globin Gene, supplied by Stratagene, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tanabe xenopus β globin gene
    Recognition of the MTRR pseudoexon with weak 5′ splice site is dependent on the presence of the c.903+469T > C mutation. A: Splicing minigene assay. Upper panel depicts the <t>β-globin</t> and HIV- tat minigenes harboring the wild-type or mutant MTRR pseudoexon. Minigenes were used to transiently transfect COS-7 cells. After RNA isolation the splicing products were analyzed by RT-PCR using minigene-specific primers. The lower bands represent correctly spliced minigene exons, the upper bands represent MTRR pseudoexon inserted between minigene exons. B: 5′ splice site optimization. The suboptimal pseudoexon 5′ splice site ( A AG/gt c ag c ) was converted to an optimal 5′ splice site (variant +3A: C AG/gt a ag t ) or to the nearly optimal 5′ splice site (variant 13G: C AG/gt g ag t ), and wild-type and mutant minigenes were analyzed by transfection/RT-PCR. The scores of the different 5′ splice sites assessed by the MaxEntScan program ( http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html ) [ Yeo and Burge, 2004 ] are shown in the left panel. Right panel shows results from RT-PCR analysis of splicing products.
    Xenopus β Globin Gene, supplied by Tanabe, 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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    85
    Roche β globin gene kit
    Recognition of the MTRR pseudoexon with weak 5′ splice site is dependent on the presence of the c.903+469T > C mutation. A: Splicing minigene assay. Upper panel depicts the <t>β-globin</t> and HIV- tat minigenes harboring the wild-type or mutant MTRR pseudoexon. Minigenes were used to transiently transfect COS-7 cells. After RNA isolation the splicing products were analyzed by RT-PCR using minigene-specific primers. The lower bands represent correctly spliced minigene exons, the upper bands represent MTRR pseudoexon inserted between minigene exons. B: 5′ splice site optimization. The suboptimal pseudoexon 5′ splice site ( A AG/gt c ag c ) was converted to an optimal 5′ splice site (variant +3A: C AG/gt a ag t ) or to the nearly optimal 5′ splice site (variant 13G: C AG/gt g ag t ), and wild-type and mutant minigenes were analyzed by transfection/RT-PCR. The scores of the different 5′ splice sites assessed by the MaxEntScan program ( http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html ) [ Yeo and Burge, 2004 ] are shown in the left panel. Right panel shows results from RT-PCR analysis of splicing products.
    β Globin Gene Kit, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    TaKaRa β globin gene 158
    Recognition of the MTRR pseudoexon with weak 5′ splice site is dependent on the presence of the c.903+469T > C mutation. A: Splicing minigene assay. Upper panel depicts the <t>β-globin</t> and HIV- tat minigenes harboring the wild-type or mutant MTRR pseudoexon. Minigenes were used to transiently transfect COS-7 cells. After RNA isolation the splicing products were analyzed by RT-PCR using minigene-specific primers. The lower bands represent correctly spliced minigene exons, the upper bands represent MTRR pseudoexon inserted between minigene exons. B: 5′ splice site optimization. The suboptimal pseudoexon 5′ splice site ( A AG/gt c ag c ) was converted to an optimal 5′ splice site (variant +3A: C AG/gt a ag t ) or to the nearly optimal 5′ splice site (variant 13G: C AG/gt g ag t ), and wild-type and mutant minigenes were analyzed by transfection/RT-PCR. The scores of the different 5′ splice sites assessed by the MaxEntScan program ( http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html ) [ Yeo and Burge, 2004 ] are shown in the left panel. Right panel shows results from RT-PCR analysis of splicing products.
    β Globin Gene 158, supplied by TaKaRa, 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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    91
    Roche beta globin gene kit
    Recognition of the MTRR pseudoexon with weak 5′ splice site is dependent on the presence of the c.903+469T > C mutation. A: Splicing minigene assay. Upper panel depicts the <t>β-globin</t> and HIV- tat minigenes harboring the wild-type or mutant MTRR pseudoexon. Minigenes were used to transiently transfect COS-7 cells. After RNA isolation the splicing products were analyzed by RT-PCR using minigene-specific primers. The lower bands represent correctly spliced minigene exons, the upper bands represent MTRR pseudoexon inserted between minigene exons. B: 5′ splice site optimization. The suboptimal pseudoexon 5′ splice site ( A AG/gt c ag c ) was converted to an optimal 5′ splice site (variant +3A: C AG/gt a ag t ) or to the nearly optimal 5′ splice site (variant 13G: C AG/gt g ag t ), and wild-type and mutant minigenes were analyzed by transfection/RT-PCR. The scores of the different 5′ splice sites assessed by the MaxEntScan program ( http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html ) [ Yeo and Burge, 2004 ] are shown in the left panel. Right panel shows results from RT-PCR analysis of splicing products.
    Beta Globin Gene Kit, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher β globin gene specific primers
    Fig. 6. The APOBEC1–ACF complex contains edited RNA. ( A ) RT–PCR products (398 bp) derived from immunoprecipitated RNA isolated from nuclear (N) and cytoplasmic (C) extracts obtained by transfecting APOBEC1, ACF or both in CCL13 cells along with the 55C <t>β-globin</t> DNA (55C). Transfected 55C plasmid containing genomic DNA generated a 1390 bp PCR product. Western blot analysis of ACF, APOBEC1 and histone H3 from identical extracts used above and the percentage of apoB RNA editing is shown. ( B ) RT–PCR (20 and 35 cycles) of input (IN), unbound (UB) and immunoprecipitated (IP) RNA from cytoplasmic extracts containing APOBEC1–ACF. The amount of product is shown as a percentage of the input RNA-derived product for each experimental condition.
    β Globin Gene Specific Primers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Illumina Inc β globin reporter gene plasmid
    Human <t>β-globin</t> reporter gene. ( A ) Detail of the reporter construct (See ‘Materials and methods’). Top schematic drawn to scale. ( B ) Imaging of gene expression from nascent transcripts (arrow) to protein product (blue channel shows CFP-SKL protein product of the reporter gene accumulating in peroxisomes). Images are maximum intensity projections of z-stacks (Δz = 0.25 μm, exposure = 1 s). ( C ) Splicing efficiency and ( D ) poly(A) tail site/length of the β-globin reporter, measured in three conditions: mock-transfected cells and cells transfected with either wild type (WT) or mutant (S34F) splicing factor U2AF1 (see Materials and methods). Error bars are SEM calculated over four measurements. ( E ) Expected fluorescence time profiles for a single transcript. When the PP7 cassette is transcribed, the red fluorescence signal increases progressively (as RNA stem loops are formed) and plateaus once the polymerase exits the cassette. The same applies to the green fluorescence signal when the PP7 cassette is transcribed. If splicing is post-release, red and green signals drop simultaneously when the unspliced RNA is released and diffuse away. If splicing is pre-release, the red fluorescence drops before the green fluorescence reflecting that intron removal occurred before the release of a spliced transcript. DOI: http://dx.doi.org/10.7554/eLife.03939.004
    β Globin Reporter Gene Plasmid, supplied by Illumina Inc, 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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    89
    Roche human β globin gene
    Localization of EJC proteins, NXF1/TAP, and p15 in the nucleus of MEL cells. ( A, a ) Schematic representation of the wild-type human <t>β-globin</t> construct. The wild-type human β-globin gene (βWT) is within the microlocus control region
    Human β Globin Gene, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher β globin housekeeping gene
    Localization of EJC proteins, NXF1/TAP, and p15 in the nucleus of MEL cells. ( A, a ) Schematic representation of the wild-type human <t>β-globin</t> construct. The wild-type human β-globin gene (βWT) is within the microlocus control region
    β Globin Housekeeping Gene, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human β globin gene
    Localization of EJC proteins, NXF1/TAP, and p15 in the nucleus of MEL cells. ( A, a ) Schematic representation of the wild-type human <t>β-globin</t> construct. The wild-type human β-globin gene (βWT) is within the microlocus control region
    Human β Globin Gene, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene human beta globin gene
    Effect of CAMs and nucleos(t)ide analogs on intracellular HBV RNA and DNA. (A and B) Uninfected and infected HepaRG cells were treated with DMSO or 30 μM BAY 41-4109_IE, BAY 41-4109, LMV, TFV, or NVR 3-1983 for 6 days prior to Northern blot analysis of intracellular ribosomal 18S and 28S RNA (A, top), HBV pgRNA and preS/S mRNA transcripts (A, middle), and encapsidated pgRNA (A, bottom) and Southern blot analysis of intracellular encapsidated HBV DNA (B). (C to F) Infected HepaRG cells were treated with increasing concentrations of NVR 3-1983 or LMV for 6 days, prior to measuring the amonts of intracellular HBV DNA (C, E) or encapsidated HBV RNA (●), total HBV mRNA transcripts (■), and <t>beta-actin</t> RNA (▲) (D, F). (G and H) Effect of 10 μM LMV or BAY 41-4109 on HBV cccDNA (G) and secreted HBsAg (H). Data points and error bars represent means and standard deviations from at least three independent studies, respectively.
    Human Beta Globin Gene, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche β globin human control gene
    Effect of CAMs and nucleos(t)ide analogs on intracellular HBV RNA and DNA. (A and B) Uninfected and infected HepaRG cells were treated with DMSO or 30 μM BAY 41-4109_IE, BAY 41-4109, LMV, TFV, or NVR 3-1983 for 6 days prior to Northern blot analysis of intracellular ribosomal 18S and 28S RNA (A, top), HBV pgRNA and preS/S mRNA transcripts (A, middle), and encapsidated pgRNA (A, bottom) and Southern blot analysis of intracellular encapsidated HBV DNA (B). (C to F) Infected HepaRG cells were treated with increasing concentrations of NVR 3-1983 or LMV for 6 days, prior to measuring the amonts of intracellular HBV DNA (C, E) or encapsidated HBV RNA (●), total HBV mRNA transcripts (■), and <t>beta-actin</t> RNA (▲) (D, F). (G and H) Effect of 10 μM LMV or BAY 41-4109 on HBV cccDNA (G) and secreted HBsAg (H). Data points and error bars represent means and standard deviations from at least three independent studies, respectively.
    β Globin Human Control Gene, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche β globin gene amplification
    Effect of CAMs and nucleos(t)ide analogs on intracellular HBV RNA and DNA. (A and B) Uninfected and infected HepaRG cells were treated with DMSO or 30 μM BAY 41-4109_IE, BAY 41-4109, LMV, TFV, or NVR 3-1983 for 6 days prior to Northern blot analysis of intracellular ribosomal 18S and 28S RNA (A, top), HBV pgRNA and preS/S mRNA transcripts (A, middle), and encapsidated pgRNA (A, bottom) and Southern blot analysis of intracellular encapsidated HBV DNA (B). (C to F) Infected HepaRG cells were treated with increasing concentrations of NVR 3-1983 or LMV for 6 days, prior to measuring the amonts of intracellular HBV DNA (C, E) or encapsidated HBV RNA (●), total HBV mRNA transcripts (■), and <t>beta-actin</t> RNA (▲) (D, F). (G and H) Effect of 10 μM LMV or BAY 41-4109 on HBV cccDNA (G) and secreted HBsAg (H). Data points and error bars represent means and standard deviations from at least three independent studies, respectively.
    β Globin Gene Amplification, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNAnexus beta globin gene
    Effect of CAMs and nucleos(t)ide analogs on intracellular HBV RNA and DNA. (A and B) Uninfected and infected HepaRG cells were treated with DMSO or 30 μM BAY 41-4109_IE, BAY 41-4109, LMV, TFV, or NVR 3-1983 for 6 days prior to Northern blot analysis of intracellular ribosomal 18S and 28S RNA (A, top), HBV pgRNA and preS/S mRNA transcripts (A, middle), and encapsidated pgRNA (A, bottom) and Southern blot analysis of intracellular encapsidated HBV DNA (B). (C to F) Infected HepaRG cells were treated with increasing concentrations of NVR 3-1983 or LMV for 6 days, prior to measuring the amonts of intracellular HBV DNA (C, E) or encapsidated HBV RNA (●), total HBV mRNA transcripts (■), and <t>beta-actin</t> RNA (▲) (D, F). (G and H) Effect of 10 μM LMV or BAY 41-4109 on HBV cccDNA (G) and secreted HBsAg (H). Data points and error bars represent means and standard deviations from at least three independent studies, respectively.
    Beta Globin Gene, supplied by DNAnexus, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Marker Gene Technologies wild type β globin gene sequence
    Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and <t>β-globin</t> gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.
    Wild Type β Globin Gene Sequence, supplied by Marker Gene Technologies, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Marker Gene Technologies β globin locus gene expression
    Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and <t>β-globin</t> gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.
    β Globin Locus Gene Expression, supplied by Marker Gene Technologies, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β globin locus gene expression/product/Marker Gene Technologies
    Average 80 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    β globin locus gene expression - by Bioz Stars, 2020-07
    80/100 stars
      Buy from Supplier

    91
    Roche β globin
    Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and <t>β-globin</t> gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.
    β Globin, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β globin/product/Roche
    Average 91 stars, based on 222 article reviews
    Price from $9.99 to $1999.99
    β globin - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    Image Search Results


    Association of TTP with ARE-containing mRNAs. ( A ) The TNFα ARE-containing β-globin reporter (TNF-UTR) or the control reporter (TNF-ΔARE), as shown in the top schematics, was transfected into HeLa cells. The cell lysates were subjected to immunoprecipitation using anti-TRN. Precipitated RNAs were analyzed by RT–PCR using primers specific to the reporters. ( B ) HeLa cells were mock-transfected or transiently transfected with siRNA against TRN. Depletion of TRN was examined by immunoblotting using anti-TRN. For mRNA stability analysis, the reporters used were the same as those in A, and meanwhile a GFP expression vector was cotransfected as a reference. Cells were harvested at the indicated time points after doxycycline addition. RNase protection was performed using 32 P-labeled probes specific to the reporter or the GFP mRNA. The graph shows the level of reporter mRNA remaining at each time point relative to that of the GFP reference. Arbitrary mRNA intensity was determined by phosphoimaging. Average and standard deviation were obtained from three independent experiments. The representative gel shows an experiment using the TNF-UTR reporter.

    Journal: Nucleic Acids Research

    Article Title: A role for transportin in deposition of TTP to cytoplasmic RNA granules and mRNA decay

    doi: 10.1093/nar/gkp717

    Figure Lengend Snippet: Association of TTP with ARE-containing mRNAs. ( A ) The TNFα ARE-containing β-globin reporter (TNF-UTR) or the control reporter (TNF-ΔARE), as shown in the top schematics, was transfected into HeLa cells. The cell lysates were subjected to immunoprecipitation using anti-TRN. Precipitated RNAs were analyzed by RT–PCR using primers specific to the reporters. ( B ) HeLa cells were mock-transfected or transiently transfected with siRNA against TRN. Depletion of TRN was examined by immunoblotting using anti-TRN. For mRNA stability analysis, the reporters used were the same as those in A, and meanwhile a GFP expression vector was cotransfected as a reference. Cells were harvested at the indicated time points after doxycycline addition. RNase protection was performed using 32 P-labeled probes specific to the reporter or the GFP mRNA. The graph shows the level of reporter mRNA remaining at each time point relative to that of the GFP reference. Arbitrary mRNA intensity was determined by phosphoimaging. Average and standard deviation were obtained from three independent experiments. The representative gel shows an experiment using the TNF-UTR reporter.

    Article Snippet: The probes used for RNase protection assays were nucleotides 307 to 1 of the GFP coding region and nucleotides 390 to 214 of the rabbit β-globin gene (accession number V00882); each corresponding DNA fragment was amplified by PCR and subcloned into pBluescript® II KS (+) (Stratagene).

    Techniques: Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Labeling, Standard Deviation

    Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and β-globin primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.

    Journal: Scientific Reports

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

    doi: 10.1038/srep05074

    Figure Lengend Snippet: Fractionation of VSV-infected 293T cells. 293T cells were infected with VSV at different MOI (0.0001 to 0.1) and incubated for 16 hr. The nuclear and cytoplasmic fractions were separated and subjected to real-time PCR using the VSV-N (left panel) and β-globin primers (right panel). The DNA copy number per cell was estimated using the VSV N gene or β-globin gene in the control plasmid and is indicated on the axis. The means ± SE of two independent experiments performed in duplicate are shown.

    Article Snippet: The copy number of each product was estimated using standard curves that were obtained using serial dilutions of the plasmids containing the VSV N and β-globin genes (pGEM®-T Easy vector) (Promega).

    Techniques: Fractionation, Infection, Incubation, Real-time Polymerase Chain Reaction, Plasmid Preparation

    LINE-1 transduction of the NP-2 cell line and its effect on VSV DNA formation. (a) LINE-1 transduction of the NP-2 cell line. The LINE-1 ORF1p and β-actin levels in the 293T, NP-2, NP-2/LINE-1 (L1.2), and NP-2/LINE-1 (L1.2 ORF2mut) cells were examined via Western blot using the anti-ORF1p IgY antibody. The ORF1p protein was observed at a molecular weight of ~41 kDa. The membrane was re-probed with an anti-β-actin antibody. The size marker at 38 kDa is indicated. Note that cropped western blots are shown and that full-length images are presented in the supplementary information . (b) The effect of LINE-1 transduction on VSV DNA production. The cells described above were infected with VSV, and the cell lysates were prepared. Real-time PCR was performed with either the N478-F/N681-R or β-globin primer pair. In addition, the VSV progeny produced by these cells were titrated and the VSV N mRNA expression levels were compared among these cells.

    Journal: Scientific Reports

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

    doi: 10.1038/srep05074

    Figure Lengend Snippet: LINE-1 transduction of the NP-2 cell line and its effect on VSV DNA formation. (a) LINE-1 transduction of the NP-2 cell line. The LINE-1 ORF1p and β-actin levels in the 293T, NP-2, NP-2/LINE-1 (L1.2), and NP-2/LINE-1 (L1.2 ORF2mut) cells were examined via Western blot using the anti-ORF1p IgY antibody. The ORF1p protein was observed at a molecular weight of ~41 kDa. The membrane was re-probed with an anti-β-actin antibody. The size marker at 38 kDa is indicated. Note that cropped western blots are shown and that full-length images are presented in the supplementary information . (b) The effect of LINE-1 transduction on VSV DNA production. The cells described above were infected with VSV, and the cell lysates were prepared. Real-time PCR was performed with either the N478-F/N681-R or β-globin primer pair. In addition, the VSV progeny produced by these cells were titrated and the VSV N mRNA expression levels were compared among these cells.

    Article Snippet: The copy number of each product was estimated using standard curves that were obtained using serial dilutions of the plasmids containing the VSV N and β-globin genes (pGEM®-T Easy vector) (Promega).

    Techniques: Transduction, Western Blot, Molecular Weight, Marker, Infection, Real-time Polymerase Chain Reaction, Produced, Expressing

    Cell-based differences in VSV DNA production. Various human cells were infected with VSV at an MOI of 0.1. The DNA was extracted 16 hr later and subjected to PCR amplification. Real-time PCR was performed using the N478-F/N681-R or β-globin primer pair. The copy numbers of the VSV N and β-globin genes in each cell line are shown in closed and open bars, respectively. The arrows indicate that the copy number per cell was below the detection limit. The positive cells are ordered according to the VSV N DNA level, followed by the VSV DNA-negative adherent cells. Subsequently, the VSV DNA-negative suspension cells are arranged in alphabetical order. The means ± SE of two independent experiments performed in duplicate are shown.

    Journal: Scientific Reports

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

    doi: 10.1038/srep05074

    Figure Lengend Snippet: Cell-based differences in VSV DNA production. Various human cells were infected with VSV at an MOI of 0.1. The DNA was extracted 16 hr later and subjected to PCR amplification. Real-time PCR was performed using the N478-F/N681-R or β-globin primer pair. The copy numbers of the VSV N and β-globin genes in each cell line are shown in closed and open bars, respectively. The arrows indicate that the copy number per cell was below the detection limit. The positive cells are ordered according to the VSV N DNA level, followed by the VSV DNA-negative adherent cells. Subsequently, the VSV DNA-negative suspension cells are arranged in alphabetical order. The means ± SE of two independent experiments performed in duplicate are shown.

    Article Snippet: The copy number of each product was estimated using standard curves that were obtained using serial dilutions of the plasmids containing the VSV N and β-globin genes (pGEM®-T Easy vector) (Promega).

    Techniques: Infection, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Recognition of the MTRR pseudoexon with weak 5′ splice site is dependent on the presence of the c.903+469T > C mutation. A: Splicing minigene assay. Upper panel depicts the β-globin and HIV- tat minigenes harboring the wild-type or mutant MTRR pseudoexon. Minigenes were used to transiently transfect COS-7 cells. After RNA isolation the splicing products were analyzed by RT-PCR using minigene-specific primers. The lower bands represent correctly spliced minigene exons, the upper bands represent MTRR pseudoexon inserted between minigene exons. B: 5′ splice site optimization. The suboptimal pseudoexon 5′ splice site ( A AG/gt c ag c ) was converted to an optimal 5′ splice site (variant +3A: C AG/gt a ag t ) or to the nearly optimal 5′ splice site (variant 13G: C AG/gt g ag t ), and wild-type and mutant minigenes were analyzed by transfection/RT-PCR. The scores of the different 5′ splice sites assessed by the MaxEntScan program ( http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html ) [ Yeo and Burge, 2004 ] are shown in the left panel. Right panel shows results from RT-PCR analysis of splicing products.

    Journal: Human Mutation

    Article Title: The Deep Intronic c.903+469T > C Mutation in the MTRR Gene Creates an SF2/ASF Binding Exonic Splicing Enhancer, Which Leads to Pseudoexon Activation and Causes the cblE Type of Homocystinuria

    doi: 10.1002/humu.21206

    Figure Lengend Snippet: Recognition of the MTRR pseudoexon with weak 5′ splice site is dependent on the presence of the c.903+469T > C mutation. A: Splicing minigene assay. Upper panel depicts the β-globin and HIV- tat minigenes harboring the wild-type or mutant MTRR pseudoexon. Minigenes were used to transiently transfect COS-7 cells. After RNA isolation the splicing products were analyzed by RT-PCR using minigene-specific primers. The lower bands represent correctly spliced minigene exons, the upper bands represent MTRR pseudoexon inserted between minigene exons. B: 5′ splice site optimization. The suboptimal pseudoexon 5′ splice site ( A AG/gt c ag c ) was converted to an optimal 5′ splice site (variant +3A: C AG/gt a ag t ) or to the nearly optimal 5′ splice site (variant 13G: C AG/gt g ag t ), and wild-type and mutant minigenes were analyzed by transfection/RT-PCR. The scores of the different 5′ splice sites assessed by the MaxEntScan program ( http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html ) [ Yeo and Burge, 2004 ] are shown in the left panel. Right panel shows results from RT-PCR analysis of splicing products.

    Article Snippet: β-Globin minigene The region of exon 1–intron 1–exon 2 of the β-globin gene was amplified by β-NotI-S (5′-ttattagcggccgcattgcttacatttgcttctga-3′) and β-HindIII-AS (5′-tgatcggttaagcttgcccttgaggttgtcca-3′) primers and ligated into pCMV-Tag-1 (Stratagene, La Jolla, CA) to produce the pCMV/β vector.

    Techniques: Mutagenesis, Mini Gene Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Transfection

    SF2/ASF regulates MTRR pseudoexon inclusion by binding to an MTRR ESE. A: SF2/ASF overexpression. HEK293 cells were cotransfected by the wild-type or c.903+469T > C mutant MTRR β-globin, and vectors expressing SF2/ASF, SRp40 or SRp55, respectively. After RNA isolation the splicing products were analyzed by RT-PCR using minigene specific primers. The lower band represents correctly spliced minigene exons, the upper band represents the MTRR pseudoexon inserted between the minigene exons. B: RNA interference. HEK-293 cells were transfected with double-stranded RNA oligonucleotides directed toward the SF2/ASF mRNA or a negative control, followed by transfection with the MTRR β-globin or HIV- tat minigenes (left panels). The degree of SF2/ASF downregulation was tested by Western blotting (right panel). The results of the Western blot is shown below the corresponding lanes.

    Journal: Human Mutation

    Article Title: The Deep Intronic c.903+469T > C Mutation in the MTRR Gene Creates an SF2/ASF Binding Exonic Splicing Enhancer, Which Leads to Pseudoexon Activation and Causes the cblE Type of Homocystinuria

    doi: 10.1002/humu.21206

    Figure Lengend Snippet: SF2/ASF regulates MTRR pseudoexon inclusion by binding to an MTRR ESE. A: SF2/ASF overexpression. HEK293 cells were cotransfected by the wild-type or c.903+469T > C mutant MTRR β-globin, and vectors expressing SF2/ASF, SRp40 or SRp55, respectively. After RNA isolation the splicing products were analyzed by RT-PCR using minigene specific primers. The lower band represents correctly spliced minigene exons, the upper band represents the MTRR pseudoexon inserted between the minigene exons. B: RNA interference. HEK-293 cells were transfected with double-stranded RNA oligonucleotides directed toward the SF2/ASF mRNA or a negative control, followed by transfection with the MTRR β-globin or HIV- tat minigenes (left panels). The degree of SF2/ASF downregulation was tested by Western blotting (right panel). The results of the Western blot is shown below the corresponding lanes.

    Article Snippet: β-Globin minigene The region of exon 1–intron 1–exon 2 of the β-globin gene was amplified by β-NotI-S (5′-ttattagcggccgcattgcttacatttgcttctga-3′) and β-HindIII-AS (5′-tgatcggttaagcttgcccttgaggttgtcca-3′) primers and ligated into pCMV-Tag-1 (Stratagene, La Jolla, CA) to produce the pCMV/β vector.

    Techniques: Binding Assay, Over Expression, Mutagenesis, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Western Blot

    Fig. 6. The APOBEC1–ACF complex contains edited RNA. ( A ) RT–PCR products (398 bp) derived from immunoprecipitated RNA isolated from nuclear (N) and cytoplasmic (C) extracts obtained by transfecting APOBEC1, ACF or both in CCL13 cells along with the 55C β-globin DNA (55C). Transfected 55C plasmid containing genomic DNA generated a 1390 bp PCR product. Western blot analysis of ACF, APOBEC1 and histone H3 from identical extracts used above and the percentage of apoB RNA editing is shown. ( B ) RT–PCR (20 and 35 cycles) of input (IN), unbound (UB) and immunoprecipitated (IP) RNA from cytoplasmic extracts containing APOBEC1–ACF. The amount of product is shown as a percentage of the input RNA-derived product for each experimental condition.

    Journal: The EMBO Journal

    Article Title: The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay

    doi: 10.1093/emboj/cdg369

    Figure Lengend Snippet: Fig. 6. The APOBEC1–ACF complex contains edited RNA. ( A ) RT–PCR products (398 bp) derived from immunoprecipitated RNA isolated from nuclear (N) and cytoplasmic (C) extracts obtained by transfecting APOBEC1, ACF or both in CCL13 cells along with the 55C β-globin DNA (55C). Transfected 55C plasmid containing genomic DNA generated a 1390 bp PCR product. Western blot analysis of ACF, APOBEC1 and histone H3 from identical extracts used above and the percentage of apoB RNA editing is shown. ( B ) RT–PCR (20 and 35 cycles) of input (IN), unbound (UB) and immunoprecipitated (IP) RNA from cytoplasmic extracts containing APOBEC1–ACF. The amount of product is shown as a percentage of the input RNA-derived product for each experimental condition.

    Article Snippet: RNA isolated from the beads with RNAzol (Biogenesis) was amplified by RT–PCR using β-globin gene-specific primers, and TOPO cloned (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Immunoprecipitation, Isolation, Transfection, Plasmid Preparation, Generated, Polymerase Chain Reaction, Western Blot

    Fig. 5. ACF protects apoB RNA from APOBEC1-induced NMD. ( A ) Northern blot analysis of total RNA from CCL13 cells transfected with 10 µg of plasmid DNA containing β-globin wild-type (wt), PTC 39, 55 or 261 nucleotides of unedited (C) and edited (T) apoB RNA inserted in-frame in exon 2 of β-globin wild type (55C, 55T, 261C and 261T) in the same position as PTC 39. Northern blots probed with β-globin cDNA. Ethidium bromide staining of the 18S RNA from the corresponding gel is shown. ( B ) Northern blot analysis of total RNA obtained from cells transfected with plasmids 55T or 55C (10 µg) together with 5 µg of lacZ and 0–10 µg of APOBEC1 in a total of 30 µg of DNA per transfection and probed with lacZ, APOBEC1 and β-globin cDNAs. In some experiments, empty vector plasmid DNA was used to make up the total concentration of DNA to 30 µg. ( C ) Northern blot analysis of total RNA as in (B) except APOBEC1 was replaced with APOBEC1(C96A) catalytically inactive mutant and probed with β-globin cDNA. Other probes are not shown. ( D ) Northern blot analysis of total RNA from cells transfected with β-globin wild-type (wt), PTC 39, 55T or 55C (10 µg) together with 5 µg of lacZ, 30 µg of ACF and 1–10 µg of APOBEC1 in a total of 55 µg of DNA per transfection, and probed with lacZ, ACF and β-globin cDNAs. Empty vector plasmid DNA was used to make up the total concentration of DNA to 55 µg. ( E ) Northern blot analysis of total RNA as in (D) except ACF was replaced with ACFΔ55 and probed as in (D). Only the β-globin probe is shown. ( F ) Northern blot analysis of total RNA from cells transfected with 55T or 55C (10 µg) together with 5 µg of lacZ, 15 µg of APOBEC1 and 10–20 µg of Upf1 wild-type (WT) or R844C dominant-negative mutant (DN) in a total of 50 µg of DNA per transfection and probed with lacZ, Upf1, APOBEC1 and β-globin cDNAs. Empty vector plasmid DNA was used to make up the total concentration of DNA to 50 µg.

    Journal: The EMBO Journal

    Article Title: The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay

    doi: 10.1093/emboj/cdg369

    Figure Lengend Snippet: Fig. 5. ACF protects apoB RNA from APOBEC1-induced NMD. ( A ) Northern blot analysis of total RNA from CCL13 cells transfected with 10 µg of plasmid DNA containing β-globin wild-type (wt), PTC 39, 55 or 261 nucleotides of unedited (C) and edited (T) apoB RNA inserted in-frame in exon 2 of β-globin wild type (55C, 55T, 261C and 261T) in the same position as PTC 39. Northern blots probed with β-globin cDNA. Ethidium bromide staining of the 18S RNA from the corresponding gel is shown. ( B ) Northern blot analysis of total RNA obtained from cells transfected with plasmids 55T or 55C (10 µg) together with 5 µg of lacZ and 0–10 µg of APOBEC1 in a total of 30 µg of DNA per transfection and probed with lacZ, APOBEC1 and β-globin cDNAs. In some experiments, empty vector plasmid DNA was used to make up the total concentration of DNA to 30 µg. ( C ) Northern blot analysis of total RNA as in (B) except APOBEC1 was replaced with APOBEC1(C96A) catalytically inactive mutant and probed with β-globin cDNA. Other probes are not shown. ( D ) Northern blot analysis of total RNA from cells transfected with β-globin wild-type (wt), PTC 39, 55T or 55C (10 µg) together with 5 µg of lacZ, 30 µg of ACF and 1–10 µg of APOBEC1 in a total of 55 µg of DNA per transfection, and probed with lacZ, ACF and β-globin cDNAs. Empty vector plasmid DNA was used to make up the total concentration of DNA to 55 µg. ( E ) Northern blot analysis of total RNA as in (D) except ACF was replaced with ACFΔ55 and probed as in (D). Only the β-globin probe is shown. ( F ) Northern blot analysis of total RNA from cells transfected with 55T or 55C (10 µg) together with 5 µg of lacZ, 15 µg of APOBEC1 and 10–20 µg of Upf1 wild-type (WT) or R844C dominant-negative mutant (DN) in a total of 50 µg of DNA per transfection and probed with lacZ, Upf1, APOBEC1 and β-globin cDNAs. Empty vector plasmid DNA was used to make up the total concentration of DNA to 50 µg.

    Article Snippet: RNA isolated from the beads with RNAzol (Biogenesis) was amplified by RT–PCR using β-globin gene-specific primers, and TOPO cloned (Invitrogen).

    Techniques: Northern Blot, Transfection, Plasmid Preparation, Staining, Concentration Assay, Mutagenesis, Dominant Negative Mutation

    Human β-globin reporter gene. ( A ) Detail of the reporter construct (See ‘Materials and methods’). Top schematic drawn to scale. ( B ) Imaging of gene expression from nascent transcripts (arrow) to protein product (blue channel shows CFP-SKL protein product of the reporter gene accumulating in peroxisomes). Images are maximum intensity projections of z-stacks (Δz = 0.25 μm, exposure = 1 s). ( C ) Splicing efficiency and ( D ) poly(A) tail site/length of the β-globin reporter, measured in three conditions: mock-transfected cells and cells transfected with either wild type (WT) or mutant (S34F) splicing factor U2AF1 (see Materials and methods). Error bars are SEM calculated over four measurements. ( E ) Expected fluorescence time profiles for a single transcript. When the PP7 cassette is transcribed, the red fluorescence signal increases progressively (as RNA stem loops are formed) and plateaus once the polymerase exits the cassette. The same applies to the green fluorescence signal when the PP7 cassette is transcribed. If splicing is post-release, red and green signals drop simultaneously when the unspliced RNA is released and diffuse away. If splicing is pre-release, the red fluorescence drops before the green fluorescence reflecting that intron removal occurred before the release of a spliced transcript. DOI: http://dx.doi.org/10.7554/eLife.03939.004

    Journal: eLife

    Article Title: Kinetic competition during the transcription cycle results in stochastic RNA processing

    doi: 10.7554/eLife.03939

    Figure Lengend Snippet: Human β-globin reporter gene. ( A ) Detail of the reporter construct (See ‘Materials and methods’). Top schematic drawn to scale. ( B ) Imaging of gene expression from nascent transcripts (arrow) to protein product (blue channel shows CFP-SKL protein product of the reporter gene accumulating in peroxisomes). Images are maximum intensity projections of z-stacks (Δz = 0.25 μm, exposure = 1 s). ( C ) Splicing efficiency and ( D ) poly(A) tail site/length of the β-globin reporter, measured in three conditions: mock-transfected cells and cells transfected with either wild type (WT) or mutant (S34F) splicing factor U2AF1 (see Materials and methods). Error bars are SEM calculated over four measurements. ( E ) Expected fluorescence time profiles for a single transcript. When the PP7 cassette is transcribed, the red fluorescence signal increases progressively (as RNA stem loops are formed) and plateaus once the polymerase exits the cassette. The same applies to the green fluorescence signal when the PP7 cassette is transcribed. If splicing is post-release, red and green signals drop simultaneously when the unspliced RNA is released and diffuse away. If splicing is pre-release, the red fluorescence drops before the green fluorescence reflecting that intron removal occurred before the release of a spliced transcript. DOI: http://dx.doi.org/10.7554/eLife.03939.004

    Article Snippet: The reads were trimmed to remove low quality sequences (Trimmomatic software) and were aligned to both the human genome (hg19) and the sequence of the β-globin reporter gene plasmid (Bowtie2 and Illumina CASAVA Eland softwares).

    Techniques: Construct, Imaging, Expressing, Transfection, Mutagenesis, Fluorescence

    Schematic of β-globin transcription cycle kinetics. Transcript synthesis and processing can occur through different pathways, the choice of which is governed by a kinetic competition between transcription and splicing. After transcription of the 3′ splice site, intron removal takes about 260 s and elongation until the end of the gene, about 55 s. Hence, splicing does not occur during elongation. The transcript is retained at the 3′end of the gene for a stochastic amount of time that can be shorter or longer than the remaining time to excise the intron. This results in two possible outcomes: either an unspliced pre-mRNA is released and then spliced very rapidly or splicing occurs while the transcript is retained on chromatin before being released. DOI: http://dx.doi.org/10.7554/eLife.03939.025

    Journal: eLife

    Article Title: Kinetic competition during the transcription cycle results in stochastic RNA processing

    doi: 10.7554/eLife.03939

    Figure Lengend Snippet: Schematic of β-globin transcription cycle kinetics. Transcript synthesis and processing can occur through different pathways, the choice of which is governed by a kinetic competition between transcription and splicing. After transcription of the 3′ splice site, intron removal takes about 260 s and elongation until the end of the gene, about 55 s. Hence, splicing does not occur during elongation. The transcript is retained at the 3′end of the gene for a stochastic amount of time that can be shorter or longer than the remaining time to excise the intron. This results in two possible outcomes: either an unspliced pre-mRNA is released and then spliced very rapidly or splicing occurs while the transcript is retained on chromatin before being released. DOI: http://dx.doi.org/10.7554/eLife.03939.025

    Article Snippet: The reads were trimmed to remove low quality sequences (Trimmomatic software) and were aligned to both the human genome (hg19) and the sequence of the β-globin reporter gene plasmid (Bowtie2 and Illumina CASAVA Eland softwares).

    Techniques:

    Integration site of the β-globin reporter and copy number analysis. ( A ) Example of an integration site of the reporter plasmid as identified by whole genome sequencing. Reads aligning to both genomic and plasmid sequences are shown at the bottom. The alignments identify the genomic position of the insertion and the location of the breaks in the plasmid. The number of repeats of the plasmid at the insertion site cannot be known from sequencing data. ( B ) Three insertion sites were identified in the cell line. ( C ) Semi-quantitative PCR was used to confirm the insertion sites and to estimate the total copy number of the integrated plasmid construct in the cell line. ( D ) Quantification of the PCR products shows, as expected, that amplicons internal to the plasmid were more amplified than the amplicons at the junctions. ( E ) Calibration curves were made by amplifying varying amounts of G-block DNA carrying the same primer pairs as used in ( C ). ( F ) Correcting the data in ( D ) with the calibration curves in ( E ) and taking the ratio of internal-to-junction PCR products yields a total copy number of 5.48 ± 1.47. Error is SEM. DOI: http://dx.doi.org/10.7554/eLife.03939.005

    Journal: eLife

    Article Title: Kinetic competition during the transcription cycle results in stochastic RNA processing

    doi: 10.7554/eLife.03939

    Figure Lengend Snippet: Integration site of the β-globin reporter and copy number analysis. ( A ) Example of an integration site of the reporter plasmid as identified by whole genome sequencing. Reads aligning to both genomic and plasmid sequences are shown at the bottom. The alignments identify the genomic position of the insertion and the location of the breaks in the plasmid. The number of repeats of the plasmid at the insertion site cannot be known from sequencing data. ( B ) Three insertion sites were identified in the cell line. ( C ) Semi-quantitative PCR was used to confirm the insertion sites and to estimate the total copy number of the integrated plasmid construct in the cell line. ( D ) Quantification of the PCR products shows, as expected, that amplicons internal to the plasmid were more amplified than the amplicons at the junctions. ( E ) Calibration curves were made by amplifying varying amounts of G-block DNA carrying the same primer pairs as used in ( C ). ( F ) Correcting the data in ( D ) with the calibration curves in ( E ) and taking the ratio of internal-to-junction PCR products yields a total copy number of 5.48 ± 1.47. Error is SEM. DOI: http://dx.doi.org/10.7554/eLife.03939.005

    Article Snippet: The reads were trimmed to remove low quality sequences (Trimmomatic software) and were aligned to both the human genome (hg19) and the sequence of the β-globin reporter gene plasmid (Bowtie2 and Illumina CASAVA Eland softwares).

    Techniques: Plasmid Preparation, Sequencing, Real-time Polymerase Chain Reaction, Construct, Polymerase Chain Reaction, Amplification, Blocking Assay

    Localization of EJC proteins, NXF1/TAP, and p15 in the nucleus of MEL cells. ( A, a ) Schematic representation of the wild-type human β-globin construct. The wild-type human β-globin gene (βWT) is within the microlocus control region

    Journal: RNA

    Article Title: In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei

    doi: 10.1261/rna.5258504

    Figure Lengend Snippet: Localization of EJC proteins, NXF1/TAP, and p15 in the nucleus of MEL cells. ( A, a ) Schematic representation of the wild-type human β-globin construct. The wild-type human β-globin gene (βWT) is within the microlocus control region

    Article Snippet: The probe used consists of a plasmid containing the genomic sequence of the human β-globin gene labeled with either digoxigenin-11-dUTP (Roche) or Cy3-AP3-dUTP (Amersham Pharmacia) by nick translation.

    Techniques: Construct

    RNase protection assays. ( A ) Schematic representation of the RNase protection probes used to analyze splicing and 3′-end cleavage of the human β-globin transcripts. The predicted RNase protection products are shown for each probe. ( B )

    Journal: RNA

    Article Title: In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei

    doi: 10.1261/rna.5258504

    Figure Lengend Snippet: RNase protection assays. ( A ) Schematic representation of the RNase protection probes used to analyze splicing and 3′-end cleavage of the human β-globin transcripts. The predicted RNase protection products are shown for each probe. ( B )

    Article Snippet: The probe used consists of a plasmid containing the genomic sequence of the human β-globin gene labeled with either digoxigenin-11-dUTP (Roche) or Cy3-AP3-dUTP (Amersham Pharmacia) by nick translation.

    Techniques:

    EJC proteins and spliceosomal snRNPs do not colocalize with mutant β-globin nascent transcripts. MELβIVSI were induced to differentiate for 3 d, hybridized with the RNA probe (green), and labeled with antibodies against SRm160 ( a ), REF

    Journal: RNA

    Article Title: In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei

    doi: 10.1261/rna.5258504

    Figure Lengend Snippet: EJC proteins and spliceosomal snRNPs do not colocalize with mutant β-globin nascent transcripts. MELβIVSI were induced to differentiate for 3 d, hybridized with the RNA probe (green), and labeled with antibodies against SRm160 ( a ), REF

    Article Snippet: The probe used consists of a plasmid containing the genomic sequence of the human β-globin gene labeled with either digoxigenin-11-dUTP (Roche) or Cy3-AP3-dUTP (Amersham Pharmacia) by nick translation.

    Techniques: Mutagenesis, Labeling

    Effect of CAMs and nucleos(t)ide analogs on intracellular HBV RNA and DNA. (A and B) Uninfected and infected HepaRG cells were treated with DMSO or 30 μM BAY 41-4109_IE, BAY 41-4109, LMV, TFV, or NVR 3-1983 for 6 days prior to Northern blot analysis of intracellular ribosomal 18S and 28S RNA (A, top), HBV pgRNA and preS/S mRNA transcripts (A, middle), and encapsidated pgRNA (A, bottom) and Southern blot analysis of intracellular encapsidated HBV DNA (B). (C to F) Infected HepaRG cells were treated with increasing concentrations of NVR 3-1983 or LMV for 6 days, prior to measuring the amonts of intracellular HBV DNA (C, E) or encapsidated HBV RNA (●), total HBV mRNA transcripts (■), and beta-actin RNA (▲) (D, F). (G and H) Effect of 10 μM LMV or BAY 41-4109 on HBV cccDNA (G) and secreted HBsAg (H). Data points and error bars represent means and standard deviations from at least three independent studies, respectively.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

    doi: 10.1128/AAC.00680-17

    Figure Lengend Snippet: Effect of CAMs and nucleos(t)ide analogs on intracellular HBV RNA and DNA. (A and B) Uninfected and infected HepaRG cells were treated with DMSO or 30 μM BAY 41-4109_IE, BAY 41-4109, LMV, TFV, or NVR 3-1983 for 6 days prior to Northern blot analysis of intracellular ribosomal 18S and 28S RNA (A, top), HBV pgRNA and preS/S mRNA transcripts (A, middle), and encapsidated pgRNA (A, bottom) and Southern blot analysis of intracellular encapsidated HBV DNA (B). (C to F) Infected HepaRG cells were treated with increasing concentrations of NVR 3-1983 or LMV for 6 days, prior to measuring the amonts of intracellular HBV DNA (C, E) or encapsidated HBV RNA (●), total HBV mRNA transcripts (■), and beta-actin RNA (▲) (D, F). (G and H) Effect of 10 μM LMV or BAY 41-4109 on HBV cccDNA (G) and secreted HBsAg (H). Data points and error bars represent means and standard deviations from at least three independent studies, respectively.

    Article Snippet: Plasmids containing either the genotype D HBV genome or the human beta-globin gene (Origene) were used to establish standard curves.

    Techniques: Infection, Northern Blot, Southern Blot

    Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and β-globin gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Engineering Globin Gene Expression

    doi: 10.1016/j.omtm.2018.12.004

    Figure Lengend Snippet: Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and β-globin gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.

    Article Snippet: In proof-of-concept studies, several laboratories demonstrated efficient HDR-mediated repair of the sickle cell mutation in human CD34+ or other hematopoietic stem and progenitor cells (HSPCs)., , Dever et al. used CRISPR/Cas9 to induce a DSB near the sickle cell mutation and an AAV vector to deliver a homologous DNA fragment that not only contained the wild-type β-globin gene sequence but also a selectable marker gene ( ).

    Techniques: Expressing, Non-Homologous End Joining, Binding Assay, Mutagenesis, Plasmid Preparation, Marker, Sequencing, Transplantation Assay

    Lentivirus Vector Employed in Globin Gene Therapy The diagram on top depicts the human β-globin gene locus, which consists of five genes (green boxes) that are expressed in a developmental stage-specific manner. High-level expression of the adult β-globin gene is mediated by the LCR HSs and the β-globin 3′ enhancer (blue ovals). Functional elements within all lentivirus vector systems include the long-terminal repeats (LTRs; gray boxes), splicing acceptor (SA) and donor (SD) sites, and the rev-responsive element (RRE), a structured RNA required for efficient viral replication. The TNS9 vector contains large segments of LCR elements HS2, HS3, and HS4, a wild-type β-globin gene, and a β-globin 3′enhancer. The HPV569 vector is similar to the TNS9 vector but contains a mutant β-globin gene that encodes a protein with a T to Q substitution at position 87. In addition, this vector contains two copies of the chicken HS4 (cHS4) insulator sequence in the LTRs. The Lenti-βAS3-FB vector is similar to HPV569 but expresses a β-globin protein with three amino acid substitutions (AS3) and contains single FB insulators in the LTRs. The GLOBE vector contains the β-globin gene and two large segments of the LCR (HS2 and HS3).

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Engineering Globin Gene Expression

    doi: 10.1016/j.omtm.2018.12.004

    Figure Lengend Snippet: Lentivirus Vector Employed in Globin Gene Therapy The diagram on top depicts the human β-globin gene locus, which consists of five genes (green boxes) that are expressed in a developmental stage-specific manner. High-level expression of the adult β-globin gene is mediated by the LCR HSs and the β-globin 3′ enhancer (blue ovals). Functional elements within all lentivirus vector systems include the long-terminal repeats (LTRs; gray boxes), splicing acceptor (SA) and donor (SD) sites, and the rev-responsive element (RRE), a structured RNA required for efficient viral replication. The TNS9 vector contains large segments of LCR elements HS2, HS3, and HS4, a wild-type β-globin gene, and a β-globin 3′enhancer. The HPV569 vector is similar to the TNS9 vector but contains a mutant β-globin gene that encodes a protein with a T to Q substitution at position 87. In addition, this vector contains two copies of the chicken HS4 (cHS4) insulator sequence in the LTRs. The Lenti-βAS3-FB vector is similar to HPV569 but expresses a β-globin protein with three amino acid substitutions (AS3) and contains single FB insulators in the LTRs. The GLOBE vector contains the β-globin gene and two large segments of the LCR (HS2 and HS3).

    Article Snippet: In proof-of-concept studies, several laboratories demonstrated efficient HDR-mediated repair of the sickle cell mutation in human CD34+ or other hematopoietic stem and progenitor cells (HSPCs)., , Dever et al. used CRISPR/Cas9 to induce a DSB near the sickle cell mutation and an AAV vector to deliver a homologous DNA fragment that not only contained the wild-type β-globin gene sequence but also a selectable marker gene ( ).

    Techniques: Plasmid Preparation, Expressing, Functional Assay, Mutagenesis, Sequencing