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Bio-Rad dna input
Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies <t>TERT</t> gene copies used to normalize for <t>DNA</t> input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.
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1) Product Images from "Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6"

Article Title: Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6

Journal: eLife

doi: 10.7554/eLife.27873

Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies TERT gene copies used to normalize for DNA input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.
Figure Legend Snippet: Measuring translocations after HBB and AAVS1 di-genic targeting. ( a ) Schematic showing the HBB gene on chromosome 11 and the AAVS1 locus on chromosome 19. The Cas9 cut sites are shown in red. One of the two possible monocentric translocations is shown. ( b ) The reference sequence of the HBB-AAVS1 translocation is shown in the top. Below are representative translocation sequences from GFP - BFP - HSPCs sorted seven days after targeting (see Figure 3e , left panel). ( c ) Representative ddPCR analyses quantifying translocations in NTC (non-template control), mock-electroporated, and GFP - BFP - cells (see Figure 3e , right panel). The reference assay quantifies TERT gene copies used to normalize for DNA input. The translocation assay probe binds 50 bp away from the junction and none of the identified translocations would therefore exclude probe binding.

Techniques Used: Sequencing, Translocation Assay, Binding Assay

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Article Snippet: .. Genotyping was performed on 5 ng of DNA input using the TaqMan® genotyping master mix on a Bio-Rad CFX384 real time PCR instrument. .. Genotyping was performed in 447 atrial fibrillation cases and 442 referents obtained from four studies (BioVU, Duke Biobank, MGH, and Penn Biobank), with genotype calls being performed by end state fluorescence after 40 cycles.

Polymerase Chain Reaction:

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Article Snippet: .. The SMN1 and SMN2 copy numbers were determined from 80 ng DNA input by digital droplet PCR (ddPCR) method and SMN1 and SMN2 copy number assays (Catalog #186‐3500 and #186‐3503, Bio‐Rad Laboratories), using droplet generation QX200™ droplet generator, C1000 Touch™ termal cycler measurements with QX100TM droplet reader and analysis with the Quantasoft™ v1.6.6 software. ..

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    Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic <t>DNA</t> from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) <t>PCR</t> analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.
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    Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic DNA from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) PCR analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.

    Journal: Nucleic Acids Research

    Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function

    doi: 10.1093/nar/gkt205

    Figure Lengend Snippet: Physical analysis of the alphoid tetO -HAC used for loading of tTS -containing cassettes. ( a ) Analysis of integrity of the alphoid tetO -HAC synthetic array after its transfer into HPRT-deficient HT1080 cells. Genomic DNA from the cells with the HAC was digested with SpeI endonuclease, separated by CHEF gel electrophoresis (range 10–70 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. Lane 1: the HAC with the loxP site in hamster CHO cells; lanes 2, 3, 4 and 5: four HAC clones with the loxP site in HPRT-deficient HT1080 cells; lane 6: the original HAC (clone AB2.218.21) generated in human cells ( 29 ). M1- Pulse Marker™ 0.1–200 kb (Sigma-Aldrich). ( b ) Mapping of the loxP site in a mega-base size alphoid DNA array. Genomic DNA from the cells possessing the original HAC was digested with PmeI endonuclease, separated by CHEF gel electrophoresis (range 200–1500 kb) and the transferred membrane was hybridized with the tetO-alphoid probe. A single 1.1-Mb fragment was detected for the original HAC (left, lane 1). Two bands of 650 and 500 kb were detected for genomic DNA from cells with the HAC bearing an inserted NBS1 gene (right, lanes 1 and 2). The size of the fragments was determined by comparison with the DNA size standard , Saccharomyces cerevisiae chromosomes. Lane M2: Yeast Chromosome PFG Marker BioLabs. ( c ) PCR analysis of clones with insertion of X3.1-I-EGFP-I into the HAC confirming restoration of the full-length HPRT gene.

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad, USA) and iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: HAC Assay, Nucleic Acid Electrophoresis, Clone Assay, Generated, Marker, DNA Array, Polymerase Chain Reaction

    Doxycycline regulated expression of the tTS-EYFP transgene loaded into the HAC. ( a ) A diagram illustrating time-course analysis of tTS-EYFP expression in the cells grown in different media. Samples 1–11 correspond to cultures used for FACS analysis. The cultures marked by red stars were analyzed by ChIP (see below). ( b ) Relative mean EYFP fluorescence determined by FACS of cells carrying the tTS-EYFP cassette (construct #4) grown in different media. Sample 1 corresponds to control HT1080 cells without the EYFP transgene (fluorescence background). Sample 2 corresponds to the cells with the HAC carrying the tTS-EYFP cassette grown in HAT and dox + medium for 30 days. Sample 3 corresponds to the cells grown for 22 days in dox + medium without selection. Sample 4 corresponds to the cells grown for 22 days without doxycycline. Sample 5 corresponds to the cells grown for a month in dox − medium with BS selection. Sample 6 corresponds to the cells grown in bsr − dox − medium. Samples 7, 8 and 9 correspond to the cells grown in dox + BS + medium for 24 h, 6 and 12 days, correspondingly. Samples 10 and 11 correspond to the cells treated either TSA or SAHA in dox + medium. Error bars, SD ( n = 3). ( c ) Transcription of Hygro , TK, EGFP and HPRT genes from the HAC. The level of the transgene transcripts from cells cultured in dox + or dox − medium was analyzed by RT–PCR. The housekeeping gene, BRCA1 , was used as an internal control. Lane 1 (dox − ) and lane 2 (dox + ) correspond to transcripts for BRCA1 . Lane 3 (dox − ) and lane 4 (dox + ) correspond to transcripts for Hygro . Lane 5 (dox − ) and lane 7 (dox + ) correspond to transcripts for TK . Lane 6 (dox − ) and lane 8 (dox + ) correspond to transcripts for HPRT . Lane 9 (dox − ) and lane 10 (dox + ) correspond to transcripts for EGFP . Lane M- GeneRuler™ 1-kb DNA ladder. ( d ) ChIP analysis of H3K4me3 chromatin in the transgene cassette of the HAC in the presence and absence of doxycycline. The cell samples used for ChIP correspond to samples 2 and 6 in Figure 6 a and b. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, corresponding endogenous pericentromeric repeats was included as a negative control. ( e ) ChIP analysis of CENP-A chromatin in the HAC in the presence and absence of doxycycline. Enrichment is shown relative to the chromosome 21 centromere.

    Journal: Nucleic Acids Research

    Article Title: Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function

    doi: 10.1093/nar/gkt205

    Figure Lengend Snippet: Doxycycline regulated expression of the tTS-EYFP transgene loaded into the HAC. ( a ) A diagram illustrating time-course analysis of tTS-EYFP expression in the cells grown in different media. Samples 1–11 correspond to cultures used for FACS analysis. The cultures marked by red stars were analyzed by ChIP (see below). ( b ) Relative mean EYFP fluorescence determined by FACS of cells carrying the tTS-EYFP cassette (construct #4) grown in different media. Sample 1 corresponds to control HT1080 cells without the EYFP transgene (fluorescence background). Sample 2 corresponds to the cells with the HAC carrying the tTS-EYFP cassette grown in HAT and dox + medium for 30 days. Sample 3 corresponds to the cells grown for 22 days in dox + medium without selection. Sample 4 corresponds to the cells grown for 22 days without doxycycline. Sample 5 corresponds to the cells grown for a month in dox − medium with BS selection. Sample 6 corresponds to the cells grown in bsr − dox − medium. Samples 7, 8 and 9 correspond to the cells grown in dox + BS + medium for 24 h, 6 and 12 days, correspondingly. Samples 10 and 11 correspond to the cells treated either TSA or SAHA in dox + medium. Error bars, SD ( n = 3). ( c ) Transcription of Hygro , TK, EGFP and HPRT genes from the HAC. The level of the transgene transcripts from cells cultured in dox + or dox − medium was analyzed by RT–PCR. The housekeeping gene, BRCA1 , was used as an internal control. Lane 1 (dox − ) and lane 2 (dox + ) correspond to transcripts for BRCA1 . Lane 3 (dox − ) and lane 4 (dox + ) correspond to transcripts for Hygro . Lane 5 (dox − ) and lane 7 (dox + ) correspond to transcripts for TK . Lane 6 (dox − ) and lane 8 (dox + ) correspond to transcripts for HPRT . Lane 9 (dox − ) and lane 10 (dox + ) correspond to transcripts for EGFP . Lane M- GeneRuler™ 1-kb DNA ladder. ( d ) ChIP analysis of H3K4me3 chromatin in the transgene cassette of the HAC in the presence and absence of doxycycline. The cell samples used for ChIP correspond to samples 2 and 6 in Figure 6 a and b. Enrichment is shown relative to the 5S rRNA control locus. Satellite 2 sequence, Sat2, corresponding endogenous pericentromeric repeats was included as a negative control. ( e ) ChIP analysis of CENP-A chromatin in the HAC in the presence and absence of doxycycline. Enrichment is shown relative to the chromosome 21 centromere.

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad, USA) and iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Expressing, HAC Assay, FACS, Chromatin Immunoprecipitation, Fluorescence, Construct, HAT Assay, Selection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Negative Control

    AMPK promoted FOXO3 binding to the Trx promoter and formation of the FOXO3/p300 transcription complex in the Trx promoter. A : Depiction of FOXO binding sites in the Trx promoter. B : AICAR increased binding of FOXO3 to the Trx promoter. HAECs were treated with AICAR and palmitic acid (PA) for 24 h. FOXO3-DNA complexes were cross-linked by formaldehyde and immunoprecipitated with anti-FOXO3 antibody. Bound FOXO3 sites in the Trx promoter were detected by qPCR and normalized with input DNA. Relative DNA was compared and expressed as the percentage of the nontreatment control subjects. Representative blots and quantitative analysis from three independent experiments are shown. Data represent the means ± SE. * P

    Journal: Diabetes

    Article Title: Activation of the AMPK-FOXO3 Pathway Reduces Fatty Acid-Induced Increase in Intracellular Reactive Oxygen Species by Upregulating Thioredoxin

    doi: 10.2337/db08-1512

    Figure Lengend Snippet: AMPK promoted FOXO3 binding to the Trx promoter and formation of the FOXO3/p300 transcription complex in the Trx promoter. A : Depiction of FOXO binding sites in the Trx promoter. B : AICAR increased binding of FOXO3 to the Trx promoter. HAECs were treated with AICAR and palmitic acid (PA) for 24 h. FOXO3-DNA complexes were cross-linked by formaldehyde and immunoprecipitated with anti-FOXO3 antibody. Bound FOXO3 sites in the Trx promoter were detected by qPCR and normalized with input DNA. Relative DNA was compared and expressed as the percentage of the nontreatment control subjects. Representative blots and quantitative analysis from three independent experiments are shown. Data represent the means ± SE. * P

    Article Snippet: The immunoprecipitated DNA and the input DNA were quantified with the qRT-PCR detection system (Bio-Rad).

    Techniques: Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Structure and expression of Rbf1. ( A ) Microarray analysis of b -dependent rbf1 expression after induction of compatible (AB31 and AB33) and incompatible (AB32 and AB34) combinations of bE and bW . Shown are the mean expression values of two biological replicates and the standard deviation (SD) ( B ) qRT-PCR analysis of rbf1 expression after induction of compatible (AB31) and incompatible (AB32) combinations of bE and bW . Samples were taken at the time-points indicated. qRT-PCR analysis was performed using the constitutively expressed ppi gene ( um03726 ) for normalization. Expression was calculated relative to the lowest expression value. Shown are mean values of two technical replicates. ( C ) Overview of primer binding sites in the rbf1 -promoter used for qChIP experiments and alignment of three putative b-binding sites ( bbs ) in the rbf1 promoter region to the bbs of lga2 and frb52 [10] , [11] . Nucleotide positions indicated are relative to the start codon. Nucleotides identical to the bbs in lga2 [10] and frb52 [11] are in bold. ( D ) qChIP analysis of bE1 binding to the rbf1 -promoter in strains AB31 and AB31bE1:3xHA 5h after induction of the bE1/bW2-heterodimer. AB31bE1:3xHA harbours a HA-tagged bE1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody. No significant enrichment was observed in control strain AB31. In AB31bE1:3xHA, the PCR-amplicon spanning bbs1 (bbs −1377 ) is significantly enriched (t-test) when compared to the amplicon spanning a control region (−2026) (p = 5.71 10 −5 ). As additional control, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of three technical replicates of three independent experiments each, and the standard deviation (SD). ( E ) Structure of the Rbf1 protein. The potential C2H2 zinc finger domain (aa 18 to 131) and a putative NLS (RHRR) (aa 95 to 98) within this domain are marked in dark grey and black, a glutamine rich sequence (aa 365 to 373) is marked in grey. The alignment shows the four C2H2 zinc finger domains; the conserved cysteine and histidine residues are in bold. ( F ) Subcellular localization of the Rbf1-3xeGFP fusion protein. Strain AB31 rbf1:3eGFP (UMS63) was induced in CM medium supplemented with 1% arabinose (CMA) for eight hours. The functional Rbf1-3xeGFP fusion protein localizes to the nucleus. Cells were stained with DAPI to visualize nuclei. Scale bar corresponds to 10 µm.

    Journal: PLoS Pathogens

    Article Title: The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis

    doi: 10.1371/journal.ppat.1001035

    Figure Lengend Snippet: Structure and expression of Rbf1. ( A ) Microarray analysis of b -dependent rbf1 expression after induction of compatible (AB31 and AB33) and incompatible (AB32 and AB34) combinations of bE and bW . Shown are the mean expression values of two biological replicates and the standard deviation (SD) ( B ) qRT-PCR analysis of rbf1 expression after induction of compatible (AB31) and incompatible (AB32) combinations of bE and bW . Samples were taken at the time-points indicated. qRT-PCR analysis was performed using the constitutively expressed ppi gene ( um03726 ) for normalization. Expression was calculated relative to the lowest expression value. Shown are mean values of two technical replicates. ( C ) Overview of primer binding sites in the rbf1 -promoter used for qChIP experiments and alignment of three putative b-binding sites ( bbs ) in the rbf1 promoter region to the bbs of lga2 and frb52 [10] , [11] . Nucleotide positions indicated are relative to the start codon. Nucleotides identical to the bbs in lga2 [10] and frb52 [11] are in bold. ( D ) qChIP analysis of bE1 binding to the rbf1 -promoter in strains AB31 and AB31bE1:3xHA 5h after induction of the bE1/bW2-heterodimer. AB31bE1:3xHA harbours a HA-tagged bE1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody. No significant enrichment was observed in control strain AB31. In AB31bE1:3xHA, the PCR-amplicon spanning bbs1 (bbs −1377 ) is significantly enriched (t-test) when compared to the amplicon spanning a control region (−2026) (p = 5.71 10 −5 ). As additional control, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of three technical replicates of three independent experiments each, and the standard deviation (SD). ( E ) Structure of the Rbf1 protein. The potential C2H2 zinc finger domain (aa 18 to 131) and a putative NLS (RHRR) (aa 95 to 98) within this domain are marked in dark grey and black, a glutamine rich sequence (aa 365 to 373) is marked in grey. The alignment shows the four C2H2 zinc finger domains; the conserved cysteine and histidine residues are in bold. ( F ) Subcellular localization of the Rbf1-3xeGFP fusion protein. Strain AB31 rbf1:3eGFP (UMS63) was induced in CM medium supplemented with 1% arabinose (CMA) for eight hours. The functional Rbf1-3xeGFP fusion protein localizes to the nucleus. Cells were stained with DAPI to visualize nuclei. Scale bar corresponds to 10 µm.

    Article Snippet: Amplicons were normalized to input DNA using the Bio-Rad IQ5 software.

    Techniques: Expressing, Microarray, Standard Deviation, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Amplification, T-Test, Sequencing, Functional Assay, Staining

    Rbf1 binds to the promoter of the Rbf1-dependently expressed dik6 gene. (A) Overview of the dik6 promoter region investigated by qChIP in strain AB31 rbf1:3xHA . Shown are the positions of the amplicons used for qChIP (numbered from 1 to 10) and the promoter truncations and internal deletions assayed in the GFP-reporter assay. (B) qChIP analysis of Rbf1-binding to the dik6 -promoter in strains AB31 and AB31 rbf1:3xHA 5h after induction of the bE1/bW2-heterodimer in CMA. AB31 rbf1:3xHA expresses an HA-tagged Rbf1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody Start and end of the amplicons are given in nucleotides (nt) relative to the start codon of dik6 . The relative positions of the amplicons are given in (A). As additional control for qChIP, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of two technical replicates of three independent experiments each, and the standard deviation (SD). Significance of the difference to values obtained for the control-amplicon located within the dik6 open reading frame (amplicon 11) was calculated using Students t-test; the respective p-values are given for AB31 rbf1:3xHA . Highest enrichment was observed for amplicons 3, 4, 5 and 6. No significant enrichment was observed in control strain AB31. (C) The dik6 promoter fragments outlined in (A) were fused to GFP as a reporter and integrated in single copy into the ip -locus of U. maydis strain CP27 ( a2 Δb::P crg1 :rbf1 ). GFP-expression was visualized microscopically 5 hours after induction of rbf1 expression in CMA medium. GFP expression declined when the promoter was truncated from 816 bp to 638 bp, and was abolished when a 298 bp promoter fragment was used. Similarly, the internal deletion Δ3 led to reduced GFP expression, while no GFP signal was detectable in the Δ 5 deletion. Scale bar = 20 µm.

    Journal: PLoS Pathogens

    Article Title: The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis

    doi: 10.1371/journal.ppat.1001035

    Figure Lengend Snippet: Rbf1 binds to the promoter of the Rbf1-dependently expressed dik6 gene. (A) Overview of the dik6 promoter region investigated by qChIP in strain AB31 rbf1:3xHA . Shown are the positions of the amplicons used for qChIP (numbered from 1 to 10) and the promoter truncations and internal deletions assayed in the GFP-reporter assay. (B) qChIP analysis of Rbf1-binding to the dik6 -promoter in strains AB31 and AB31 rbf1:3xHA 5h after induction of the bE1/bW2-heterodimer in CMA. AB31 rbf1:3xHA expresses an HA-tagged Rbf1 protein used for immunoprecipitation with anti-HA-antibody. Numbers give the enrichment in % of the input-DNA of the PCR amplicons in DNA co-immunoprecipitated with HA-antibody Start and end of the amplicons are given in nucleotides (nt) relative to the start codon of dik6 . The relative positions of the amplicons are given in (A). As additional control for qChIP, a region from the eIF2b gene ( um04869 ) was used. Given are the mean values of two technical replicates of three independent experiments each, and the standard deviation (SD). Significance of the difference to values obtained for the control-amplicon located within the dik6 open reading frame (amplicon 11) was calculated using Students t-test; the respective p-values are given for AB31 rbf1:3xHA . Highest enrichment was observed for amplicons 3, 4, 5 and 6. No significant enrichment was observed in control strain AB31. (C) The dik6 promoter fragments outlined in (A) were fused to GFP as a reporter and integrated in single copy into the ip -locus of U. maydis strain CP27 ( a2 Δb::P crg1 :rbf1 ). GFP-expression was visualized microscopically 5 hours after induction of rbf1 expression in CMA medium. GFP expression declined when the promoter was truncated from 816 bp to 638 bp, and was abolished when a 298 bp promoter fragment was used. Similarly, the internal deletion Δ3 led to reduced GFP expression, while no GFP signal was detectable in the Δ 5 deletion. Scale bar = 20 µm.

    Article Snippet: Amplicons were normalized to input DNA using the Bio-Rad IQ5 software.

    Techniques: Reporter Assay, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Standard Deviation, Amplification, Expressing

    Loading of the tTA VP64 - containing construct into the alphoid tetO -HAC propagated in human HPRT-minus HT1080 cells. ( a ) Diagram of the construct tetR-VP64-IRES-DsRed2 used in this study. The construct contains a 3′ HPRT sequence and loxP site, the tTA VP64 (tetR with the VP64 carrying four copies of VP16 activation domain) that is co-transcribed with the DsRed2 transgene under the same CAG promoter. The cHS4 insulator ( 54 ) flanks the expressing cassette from both sides. (A similar construct, tetR*-VP16-IRES-DsRed2, carrying one copy of the VP16 domain is described in Supplementary Figure S2). Both constructs were inserted into the loxP site of the alphoid tetO -HAC propagated in HPRT-deficient HT1080 cells. ( b ) Loading of the vector into the loxP site of the HAC is accompanied by reconstitution of the HPRT gene allowing cell selection on HAT medium. ( c ) A map of the resulting transgene cluster in the HAC containing TK and Hygro genes, the DsRed2 color marker and the reconstructed HPRT gene. Arrows indicate direction of transcription of the transgenes and 30–40 copies of BS incorporated in the alphoid tetO -HAC. ( d ) Lanes 1, 2 and 3 correspond to PCR products obtained with the genomic DNA isolated from HAC-containing clones of HT1080 cells using specific primers for the HPRT gene. The PCR products were sequenced and confirmed reconstitution of the HPRT gene. M-ladder marker. ( e – g ) FISH analysis of the HAC-containing HT1080 clone. Chromosomal DNA was counterstained with DAPI (blue) (e). The HAC was visualized using a BAC32–2-mer(tetO) probe (red) (f and g).

    Journal: Nucleic Acids Research

    Article Title: Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette

    doi: 10.1093/nar/gkv124

    Figure Lengend Snippet: Loading of the tTA VP64 - containing construct into the alphoid tetO -HAC propagated in human HPRT-minus HT1080 cells. ( a ) Diagram of the construct tetR-VP64-IRES-DsRed2 used in this study. The construct contains a 3′ HPRT sequence and loxP site, the tTA VP64 (tetR with the VP64 carrying four copies of VP16 activation domain) that is co-transcribed with the DsRed2 transgene under the same CAG promoter. The cHS4 insulator ( 54 ) flanks the expressing cassette from both sides. (A similar construct, tetR*-VP16-IRES-DsRed2, carrying one copy of the VP16 domain is described in Supplementary Figure S2). Both constructs were inserted into the loxP site of the alphoid tetO -HAC propagated in HPRT-deficient HT1080 cells. ( b ) Loading of the vector into the loxP site of the HAC is accompanied by reconstitution of the HPRT gene allowing cell selection on HAT medium. ( c ) A map of the resulting transgene cluster in the HAC containing TK and Hygro genes, the DsRed2 color marker and the reconstructed HPRT gene. Arrows indicate direction of transcription of the transgenes and 30–40 copies of BS incorporated in the alphoid tetO -HAC. ( d ) Lanes 1, 2 and 3 correspond to PCR products obtained with the genomic DNA isolated from HAC-containing clones of HT1080 cells using specific primers for the HPRT gene. The PCR products were sequenced and confirmed reconstitution of the HPRT gene. M-ladder marker. ( e – g ) FISH analysis of the HAC-containing HT1080 clone. Chromosomal DNA was counterstained with DAPI (blue) (e). The HAC was visualized using a BAC32–2-mer(tetO) probe (red) (f and g).

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad).

    Techniques: Construct, HAC Assay, Sequencing, Activation Assay, Expressing, Plasmid Preparation, Selection, HAT Assay, Chromosome Transmission Fidelity Colony Color Assay, Polymerase Chain Reaction, Isolation, Clone Assay, Marker, Fluorescence In Situ Hybridization

    Chromatin/transcription changes in the alphoid tetO -HAC kinetochore induced by tTA VP64 tethering. ( a and b ) De-repression of tetO-alpha-satellite DNA transcription in the HAC when cells are grown in the medium lacking doxycycline. Quantitative RT-PCR experiments for two clones, clone #1 and clone #2, showing that the transcripts of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC are significantly repressed in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline. ( c and d ) Chromatin immunoprecipitation (ChIP) analysis of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC in two clones using antibodies against H3K4me3 in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline after 1 and 3 days of culture. Data were normalized to the internal 5S rDNA controls. ( e ) ChIP analysis of CENP-A chromatin in the alphoid tetO -HAC in the clone #2 cultured in the presence and absence of doxycycline for different time intervals (3, 4, 8 and 12 days of culture). Enrichment is shown relative to the chromosome 21 centromere. Error bars indicate s.d. The significant differences were calculated using Student's two-tailed t -test.

    Journal: Nucleic Acids Research

    Article Title: Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette

    doi: 10.1093/nar/gkv124

    Figure Lengend Snippet: Chromatin/transcription changes in the alphoid tetO -HAC kinetochore induced by tTA VP64 tethering. ( a and b ) De-repression of tetO-alpha-satellite DNA transcription in the HAC when cells are grown in the medium lacking doxycycline. Quantitative RT-PCR experiments for two clones, clone #1 and clone #2, showing that the transcripts of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC are significantly repressed in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline. ( c and d ) Chromatin immunoprecipitation (ChIP) analysis of non-coding tetO-alpha-satellite DNA repeats in the alphoid tetO -HAC in two clones using antibodies against H3K4me3 in the cells growing in doxycycline-containing medium compared to that of the cells that have been grown in the medium lacking doxycycline after 1 and 3 days of culture. Data were normalized to the internal 5S rDNA controls. ( e ) ChIP analysis of CENP-A chromatin in the alphoid tetO -HAC in the clone #2 cultured in the presence and absence of doxycycline for different time intervals (3, 4, 8 and 12 days of culture). Enrichment is shown relative to the chromosome 21 centromere. Error bars indicate s.d. The significant differences were calculated using Student's two-tailed t -test.

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad).

    Techniques: HAC Assay, Quantitative RT-PCR, Clone Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test