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  • 97
    Thermo Fisher plasmid dna
    Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 50803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore plasmid dna
    Plasmid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega plasmid dna
    a) EPR Spectra of Cr V obtained during the NADPH-dependent Cr VI reduction catalyzed by PLs (37° under room air) in the presence ( left ) vs. absence ( right ) of <t>pGEM-</t> 7Zf(+) plasmid <t>DNA</t> . The Cr V EPR signal intensity ( b ) and the decline in Cr VI ( c )
    Plasmid Dna, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 16186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad plasmid dna
    Double stranded <t>DNA</t> end preferences of SXT-Exo . SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear <t>dsDNA</t> with 4 nt 3'-overhangs (PstI-linearized pUC18, black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.
    Plasmid Dna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 6851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen dna mini kit
    Double stranded <t>DNA</t> end preferences of SXT-Exo . SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear <t>dsDNA</t> with 4 nt 3'-overhangs (PstI-linearized pUC18, black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.
    Dna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    MACHEREY NAGEL plasmid dna
    Inhibition of R.PabI activities by methylation. ( A ) Inhibition of strand cleavage. Plasmid pBAD30_ cviQIM (Supplementary Figure S1) with a gene for M.CviQI, which generates 5′-GTm6AC as M.PabI does, under control of the pBAD promoter was prepared from cultures with varying concentrations of arabinose, its inducer. After incubation with R.PabI at 85°C for 6 h, the plasmid DNAs were subjected to 0.8% agarose gel electrophoresis. OC, open circle; SC, supercoiled; P, product <t>DNA.</t> Left lane: 1 kb DNA Ladder. ( B ) Strand-specific inhibition of cleavage in hemimethylated double-stranded DNA. A 40-mer single-stranded (GTAC40T or GTAC40Tme, Supplementary Table S2) or double-stranded (GTAC40_hemi_met or GTAC40, Supplementary Table S2) substrate (1 pmol, 100 nM) with a 32 P-label (black dot) at the 5′-end of either strand was incubated with R.PabI (9.2 pmol, 920 nM) at 85°C for 3 h. Products were separated by 10% denaturing PAGE. Box, recognition sequence (5′-GTAC). Me diamond, methylation of the top strand. Cleavage at the recognition sequence resulted in 27-mer and 13-mer oligonucleotides. The supershifted bands near the top of the gel are likely DNA–R.PabI complexes (see also Figure 5 and related text). ( C ) Inhibition of DNA glycosylase. A 40-mer double-stranded substrate (GTAC40 or GTAC40_full_met (Supplementary Table S2), 1 pmol, 100 nM) with a 32 P -label at the 5′ end of both strands was incubated with R.PabI (9.2 pmol, 920 nM) and then treated with 0.1 M DMED at <t>37°C</t> for 1 h. Products were separated by 10% denaturing PAGE.
    Plasmid Dna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 94/100, based on 3446 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene plasmid dna
    The presence of exon 5 does not affect protein degradation but exhibits a profound impact on mA3 protein synthesis. (A) 293T cells were transfected with pFLAG-CMV2- mA3 d or pFLAG-CMV2- mA3 d Δ5 along with pFLAG-CMV2-GFP, which expresses green fluorescent protein (GFP) as a loading control. The cells were treated with cycloheximide for the indicated duration to stop protein synthesis and then harvested. DMSO was used as a solvent control. The FLAG-tagged mA3 and GFP were detected with the anti-FLAG antibody in immunoblotting. Endogenous IκBα expression was used as a positive control to confirm the effect of cycloheximide. (B) In vitro transcription and translation assays. <t>DNA</t> templates containing the entire coding region of the <t>BALB/c</t> full-length or Δ5 mA3 cDNA with the T7 promoter and His-tag sequence were subjected to an in vitro transcription/translation reaction by incubating for 30 or 60 min. The levels of mA3 protein synthesis and mRNA expression were evaluated by immunoblotting using the anti-His antibody and RT-PCR using the primer set a-b, respectively. Intensities of protein bands on the immunoblot membrane and DNA bands after the RT-PCR reaction and electrophoresis were measured by densitometry and are shown below each corresponding band. *, signals below detection limits.
    Plasmid Dna, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 1911 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Amaxa plasmid dna
    Characterization of the late-promoter region of HPV18. (A) The strong activity of a P 811 promoter region is dependent on CCW orientation of the HPV18 Ori. HPV18-infected human foreskin keratinocytes (HFK18), differentiated by adding 2 mM calcium, were transfected for 48 h with each of the indicated plasmids. (A) Renilla luciferase plasmid pRL-TS was <t>cotransfected</t> as an internal control. The supernatant of the cell lysates from each transfection was examined for dual luciferase activities. Relative promoter activity levels were calculated by dividing the value representing the light unit readings from a testing promoter- firefly luciferase reporter by the value representing the light unit readings from the Renilla luciferase reporter (left panel). Plasmid pXHW61 directly derived from pXHW21 has the HPV18 Ori flipped into a CCW orientation (middle panel). Plasmid pXHW21-derived pXHW49 and pXHW22-derived pXHW50 have their corresponding promoter regions replaced by the SV40 early promoter derived from the pGL3 control vector (right panel). The data shown are means ± standard deviations (SD) of results from two to three independent experiments. P values were calculated using Student’s t test. (B) The P 811 promoter activity depends on keratinocyte differentiation. HFK18 cells with (+) or without (−) 2.0 mM calcium treatment were transfected with pXHW22 for 48 h and then analyzed for their luciferase activity. The data shown are from results from one of two experiments, with means ± SD calculated from triplicate samples. (C) Opposite orientations of the HPV18 Ori do not affect plasmid <t>DNA</t> replication in HFK18 cells. HPV18-infected HFK cells, differentiated by 2 mM calcium, were transfected with pXHW21 (Ori in CW orientation) or pXHW22 (Ori in CCW orientation) for 48 h. Replicated plasmid DNA isolated from the cells and the original input bacterial plasmid DNA were compared for their sensitivity to DpnI (digesting only methylated bacterial plasmid DNA) and MboI (digesting only unmethylated bacterial and replicated plasmid DNA). Because the input bacterial DNA is methylated at the adenine of GATC sequences, it is sensitive to digestion by DpnI but resistant to digestion by MboI (right panel), and because human cells lack adenine methylase activity, the newly replicated DNA lacking adenine methylation is thus resistant to DpnI digestion but susceptible to MboI digestion (left panel). ND, no digestion with a restriction enzyme. The digested DNA samples were then resolved in a 1% agarose gel and imaged by ethidium bromide staining. Lanes 1 and 8 represent DNA markers (M). (D) Aphidicolin, a DNA polymerase inhibitor, blocks CCW orientation-dependent HPV18 late promoter activity. The sensitivity of Ori-directed DNA replication and HPV18 late promoter activity in plasmid pXHW22 to aphidicolin at different doses was analyzed in the HFK18 cells cotransfected with plasmids pXHW22 and pRL-SV40 (an internal control). The supernatant of the cell lysates was examined for dual luciferase activities, and the relative promoter activity levels were calculated as described for panel A.
    Plasmid Dna, supplied by Amaxa, used in various techniques. Bioz Stars score: 94/100, based on 1772 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Mirus Bio plasmid dna
    Transfection efficiency of polycation lipid <t>nanocarrier/DNA</t> complexes <t>(PDC)</t> (N/P = 10) in human lung adenocarcinoma (SPC-A1) cells compared with those of Lipofectamine™ 2000/DNA complexes (LDC). ( A ) Transfection efficiency of plasmid pEGFP-N2 determined by flow cytometer; ( B ) relative luciferase activity in SPC-A1 cells treated with PDC in comparison with that of LDC; ( C ) the cell viability of PDCs in SPC-A1 cells compared with that of polyethylenimine (PEI)/DNA complexes at various N/P ratios (* P
    Plasmid Dna, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 94/100, based on 1603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM plasmid dna
    Liver distribution of pMMP13 in <t>PEI</t> or HA/PEI complexes . Mice were intravenously injected with pMMP13 (1 mg/kg) in PEI complexes or HA-shielded PEI complexes. At 4 hours after injection, gDNA was extracted from blood and liver tissues. pMMP13 copy numbers in the liver were divided by those in the blood. The data are the mean ± SE ( n = 5). gDNA, genomic <t>DNA;</t> HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.
    Plasmid Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Polyplus Transfection plasmid dna
    Generation of ΔN and ΔNC mice Diagrammatic representation of ΔN ( a ) and ΔNC ( b ) knock-in mouse line generation. The endogenous <t>Mecp2</t> allele was targeted in male ES cells. The site of Cas9 cleavage in the WT sequence is shown by the scissors symbol (used for production of ΔN knock-in ES cells). The selection cassette was removed in vivo by crossing chimaeras with deleter ( CMV-Cre ) transgenic mice. Southern blot analysis shows correct targeting of ES cells and successful cassette deletion in the knock-in mice. The solid black line represents the sequence encoded in the targeted vector and the dotted lines indicate the flanking regions of mouse genomic <t>DNA.</t> For gel source data, see Supplementary Information .
    Plasmid Dna, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 93/100, based on 816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Polysciences inc plasmid dna
    Characterization of the <t>FISC</t> system. a Cre-catalyzed <t>DNA</t> recombination with FISC system using fluorescence imaging and flow cytometry. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169 (P hCMV -CreN59-L9-Coh2-NES-pA), pXY177 (P FRLd -NLS-DocS-L9-CreC60-pA), and pDL78 (P hCMV - loxP -STOP- loxP -EGFP-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) each day for 2 days, and expression of the reporter protein EGFP was visualized by fluorescence microscopy and by flow cytometry at 48 h after the first illumination. Representative images from n = 5 biological replicates. Scale bar, 250 μm. Graph bars represent mean ± SD of n = 3 biological replicates. b Cre-catalyzed DNA recombination with FISC system using luciferase assay. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169, pXY177, and pXY185 (P hCMV - loxP -STOP- loxP -Luciferase-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated as described in ( a ), and bioluminescence measurements were taken at 48 h after the first illumination ( n = 3 independent experiments). c Assessment of illumination-intensity-dependent FISC system activity. 6 × 10 4 HEK-293 cells were cotransfected with pXY137, pXY169, pXY177, and pGY125 (SEAP reporter plasmid) at a 10:1:1:20 (w/w/w/w) ratio and illuminated with FRL for 6 h each day for 2 days at eight different light intensities (0–5 mW cm −2 ); SEAP levels were profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. d Exposure-time-dependent FISC system activity. With the same plasmid ratios as in ( c ), we illuminated transfected cells with FRL (1.5 mW cm −2 , 730 nm) for different time periods (0–120 h). SEAP expression was profiled in the cell culture supernatant at 120 h after initial illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. e FISC-induced SEAP expression in multiple mammalian cell lines. Four different mammalian cell lines were cotransfected and illuminated as described in ( c ), and SEAP expression in the culture supernatant was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Evaluation of the spatial resolution for FISC-mediated transgene expression. A monolayer comprising HEK-293 cells was cotransfected with pXY137, pXY169, pXY177, and pDL78 (EGFP reporter plasmid) at a ratio of a 10:1:1:20 (w/w/w/w), and illuminated as described in ( a ), but through a photomask (schematic, left) with a 6.5 mm slit, and fluorescence microscopy based analysis of the corresponding pattern of EGFP expression at 48 h after the first illumination (right). Representative images from n = 2 biological replicates. b – e Data represent the mean ± SD. Source data for this figure are available in the Source data file.
    Plasmid Dna, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 92/100, based on 883 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Eppendorf AG plasmid dna
    Plasmid protospacer specificity of <t>AceCas9.</t> 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of <t>DNA</t> cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.
    Plasmid Dna, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 92/100, based on 1102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc plasmid dna
    Design of experiments (DOE) model number 2 for <t>NGS</t> plasmid <t>DNA</t> library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.
    Plasmid Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 632 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc plasmid dna
    Analyses of the optimized peptide sequence (Opt-pep) ( A ) Schematic illustration of <t>biotin–NCX1(301–320)</t> (native sequence), biotin–NCX1(Opt-pep) (optimized peptide sequence), biotin–NCX1(301–320) scrambled (control sequence) and an untagged PLM cyt peptide used in pull-down assay in ( B ). ( B ) Pull-down assays with biotin–NCX1(301–320) and biotin–NCX1(Opt-pep) against the untagged PLM cyt peptide. PLM binding was analysed by immunoblotting using anti-PLM. A biotin–NCX1(301–320) scrambled peptide and beads were used as negative controls. ( C ) Binding of biotin–NCX1(301–320) and biotin–NCX1(Opt-pep) was identified by overlaying the peptides on membranes containing 20-mer overlapping pSer 68 -PLM peptides, followed by immunoblotting using HRP-conjugated anti-biotin. Binding of ( D ) biotin–NCX1(235–254) and ( E ) biotin–NCX1(301–320) to pSer 68 -PLM with or without a pre-incubation of Opt-pep. Binding was analysed by immunoblotting using HRP-conjugated anti-biotin. Phosphorylated Ser 68 is underlined and the C-terminal α-helical region is indicated in ( C )–( E ). Incubation with only HRP-conjugated anti-biotin (omitting incubation with the peptides) was used as negative control (right-hand panels in C – E ). ( F ) Alignment of human, rat, mouse and dog NCX1 sequence. Position of the XIP region and the native NCX1 sequence (amino acids 301–320) used for optimization are underlined. Black boxes indicate identical amino acids <t>(DNA</t> Star).
    Plasmid Dna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 601 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim plasmid dna
    Association of <t>core-GFP</t> mutant derivatives with U17 snoRNP. HeLa cells were transfected with plasmids expressing either core-GFP, or Δ3N, Δ9C, Δ13C, and Δ20C deletion mutants. Proteins extracts were separated by SDS-PAGE and analyzed by Western blotting using anti-GFP MAbs (A) or they were incubated with anti-GFP beads and immunoprecipitated RNAs were analyzed by an RNase A/T1 protection assay using a U17-specific RNA probe (B). Protein and <t>DNA</t> size markers are indicated on the left.
    Plasmid Dna, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 637 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mission plko 1 puro non mammalian shrna control plasmid dna
    Association of <t>core-GFP</t> mutant derivatives with U17 snoRNP. HeLa cells were transfected with plasmids expressing either core-GFP, or Δ3N, Δ9C, Δ13C, and Δ20C deletion mutants. Proteins extracts were separated by SDS-PAGE and analyzed by Western blotting using anti-GFP MAbs (A) or they were incubated with anti-GFP beads and immunoprecipitated RNAs were analyzed by an RNase A/T1 protection assay using a U17-specific RNA probe (B). Protein and <t>DNA</t> size markers are indicated on the left.
    Mission Plko 1 Puro Non Mammalian Shrna Control Plasmid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene plasmid dna
    Evaluation of <t>DNA</t> methylation status at a specific sortilin 1 ( <t>SORT1</t> ) promoter CpG site as an moyamoya disease (MMD) biomarker. (A) DNA methylation status at the specific promoter CpG sites for five candidate genes was determined using pyrosequencing analysis of endothelial colony forming cells (ECFCs) from eight patients with MMD and eight normal controls. Lines inside graphs represent medians. Statistical analyses were performed using the Mann-Whitney U test. DNA methylation status at specific SORT1 CpG sites was determined using ECFC pyrosequencing analysis from an independent sample set of patients with MMD (n=7) and normal controls (n=6). (B) Receiver operating characteristic curves reveal discrimination of patients with MMD from normal controls based on DNA methylation status at a specific SORT1 promoter CpG site. APOD , apolipoprotein D; FAP , fibroblast activation protein alpha; LITAF , lipopolysacchride induced tumor necrosis factor-alpha factor; NUPR1 , nuclear protein 1; AUC, area under the curve. * P
    Plasmid Dna, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genomed GmbH plasmid dna
    Evaluation of <t>DNA</t> methylation status at a specific sortilin 1 ( <t>SORT1</t> ) promoter CpG site as an moyamoya disease (MMD) biomarker. (A) DNA methylation status at the specific promoter CpG sites for five candidate genes was determined using pyrosequencing analysis of endothelial colony forming cells (ECFCs) from eight patients with MMD and eight normal controls. Lines inside graphs represent medians. Statistical analyses were performed using the Mann-Whitney U test. DNA methylation status at specific SORT1 CpG sites was determined using ECFC pyrosequencing analysis from an independent sample set of patients with MMD (n=7) and normal controls (n=6). (B) Receiver operating characteristic curves reveal discrimination of patients with MMD from normal controls based on DNA methylation status at a specific SORT1 promoter CpG site. APOD , apolipoprotein D; FAP , fibroblast activation protein alpha; LITAF , lipopolysacchride induced tumor necrosis factor-alpha factor; NUPR1 , nuclear protein 1; AUC, area under the curve. * P
    Plasmid Dna, supplied by Genomed GmbH, used in various techniques. Bioz Stars score: 93/100, based on 616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co plasmid dna
    Genotypes of TaCRT - D in partial lines of RIL population using <t>PCR-RFLP.</t> M: <t>DNA</t> ladder 100; O: Opata 85; W: W7984; 1∼26: Partial lines of RIL population.
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    Schematic diagram depicting a restriction map of a 5.6-kb <t>HindIII</t> fragment of E. faecium JS79 <t>DNA</t> containing two streptogramin A resistance genes and a transposase gene. Bold arrows reflect the orientations of vgaD and vatG and the transposase (IS); black
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    Schematic diagram depicting a restriction map of a 5.6-kb <t>HindIII</t> fragment of E. faecium JS79 <t>DNA</t> containing two streptogramin A resistance genes and a transposase gene. Bold arrows reflect the orientations of vgaD and vatG and the transposase (IS); black
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    The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. <t>DNA</t> was sequenced using <t>M13</t> primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).
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    Image Search Results


    a) EPR Spectra of Cr V obtained during the NADPH-dependent Cr VI reduction catalyzed by PLs (37° under room air) in the presence ( left ) vs. absence ( right ) of pGEM- 7Zf(+) plasmid DNA . The Cr V EPR signal intensity ( b ) and the decline in Cr VI ( c )

    Journal:

    Article Title: Addition of DNA to CrVI and Cytochrome b5 Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and CrIII Complexes

    doi: 10.1002/cbdv.200890143

    Figure Lengend Snippet: a) EPR Spectra of Cr V obtained during the NADPH-dependent Cr VI reduction catalyzed by PLs (37° under room air) in the presence ( left ) vs. absence ( right ) of pGEM- 7Zf(+) plasmid DNA . The Cr V EPR signal intensity ( b ) and the decline in Cr VI ( c )

    Article Snippet: Plasmid DNA ( pGEM ®-7Zf(+)) was purchased from Promega (Madison, WI); seakem LE agarose was from Cambrex , and ethidium bromide was from EMD Chemicals (Germany).

    Techniques: Electron Paramagnetic Resonance, Plasmid Preparation

    DNA Strand breaks in pGEM-7Zf(+)during NADPH-dependent Cr VI reduction by the PLs

    Journal:

    Article Title: Addition of DNA to CrVI and Cytochrome b5 Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and CrIII Complexes

    doi: 10.1002/cbdv.200890143

    Figure Lengend Snippet: DNA Strand breaks in pGEM-7Zf(+)during NADPH-dependent Cr VI reduction by the PLs

    Article Snippet: Plasmid DNA ( pGEM ®-7Zf(+)) was purchased from Promega (Madison, WI); seakem LE agarose was from Cambrex , and ethidium bromide was from EMD Chemicals (Germany).

    Techniques:

    Double stranded DNA end preferences of SXT-Exo . SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear dsDNA with 4 nt 3'-overhangs (PstI-linearized pUC18, black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Double stranded DNA end preferences of SXT-Exo . SXT-Exo (2 pmol of trimers) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl; was incubated at 37°C for 30 mins with: i) 5'-phosphorylated linear dsDNA with 4 nt 3'-overhangs (PstI-linearized pUC18, black shaded circles); ii) 5'-hydroxylated linear dsDNA with 4 nt 3'-overhangs (dephosphorylated PstI-linerarized pUC18, un-shaded circles); iii) 5'-phosphorylated blunt-ended dsDNA (SspI-linerized pUC18, shaded inverted triangles); or iv) 5'-phosphorylated linear dsDNA with 4 nt 5'-overhangs (BamHI-linearized pUC18, un-shaded green triangles). Aliquots were removed at 1, 2, 5, 10, 20 and 40 minutes; quenched, then dsDNA levels were quantified using the PicoGreen reagent. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Incubation, Standard Deviation

    Digestion of fluorescently-labeled annealed oligonucleotide substrates by lambda-Exo . In experiments analogous to those described for SXT-Exo (see Figure 7), the ability of lambda-Exo to digest three different (partially) dsDNA substrates was investigated. In each assay, lambda-Exo (3 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 5 mM MgCl 2 . Aliquots were removed and quenched at 0, 0.5, 1, 2, 4 and 10 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A : Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate by SXT-Exo. Panel B : Representative gel image showing digestion of the Blunt ended DNA substrate by lambda-Exo. Panel C : Representative gel image showing digestion of the 3'-overhang DNA substrate by lambda-Exo. Panel D : Plot showing the digestion of the three DNA substrates by lambda-Exo over a 10 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Digestion of fluorescently-labeled annealed oligonucleotide substrates by lambda-Exo . In experiments analogous to those described for SXT-Exo (see Figure 7), the ability of lambda-Exo to digest three different (partially) dsDNA substrates was investigated. In each assay, lambda-Exo (3 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 5 mM MgCl 2 . Aliquots were removed and quenched at 0, 0.5, 1, 2, 4 and 10 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A : Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate by SXT-Exo. Panel B : Representative gel image showing digestion of the Blunt ended DNA substrate by lambda-Exo. Panel C : Representative gel image showing digestion of the 3'-overhang DNA substrate by lambda-Exo. Panel D : Plot showing the digestion of the three DNA substrates by lambda-Exo over a 10 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Labeling, Incubation, Fluorescence, Standard Deviation

    Efficiency of SXT-Bet + SXT-Exo mediated recombination between a PCR-generated DNA fragment and its homologous target on the E. coli chromosome . Panel A: Schematic overview of the chromosomal targeting assay use to score exonuclease + SSAP-mediate recombination efficiency. A dsDNA 'targeting' molecule ( galK

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Efficiency of SXT-Bet + SXT-Exo mediated recombination between a PCR-generated DNA fragment and its homologous target on the E. coli chromosome . Panel A: Schematic overview of the chromosomal targeting assay use to score exonuclease + SSAP-mediate recombination efficiency. A dsDNA 'targeting' molecule ( galK

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Polymerase Chain Reaction, Generated

    Digestion of fluorescently-labeled annealed oligonucleotide substrates by SXT-Exo . The ability of SXT-Exo to digest three different (partially) dsDNA substrates (prepared from two annealed oligonucleotides, see Additional File 4 ) was monitored by quantifying the digestion of the 5'-phosphorylated-3'-Cy3-labeled strand using fluorescence gel scanning. In each assay, SXT-Exo (50 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 0.5 mM MnCl 2 . Aliquots were removed and quenched at 0, 1, 2, 4, 10 and 20 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A : Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate (annealed 5'-PO 4 -70Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel B : Representative gel image showing digestion of the Blunt ended DNA substrate (annealed 5'-PO 4 -50Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel C : Representative gel image showing digestion of the 3'-overhang DNA substrate (annealed 5'-PO 4 -50Cy3 + 70overhang oligonucleotides) by SXT-Exo. Panel D : Plot showing the digestion of the three DNA substrates by SXT-Exo over a 20 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Digestion of fluorescently-labeled annealed oligonucleotide substrates by SXT-Exo . The ability of SXT-Exo to digest three different (partially) dsDNA substrates (prepared from two annealed oligonucleotides, see Additional File 4 ) was monitored by quantifying the digestion of the 5'-phosphorylated-3'-Cy3-labeled strand using fluorescence gel scanning. In each assay, SXT-Exo (50 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 0.5 mM MnCl 2 . Aliquots were removed and quenched at 0, 1, 2, 4, 10 and 20 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A : Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate (annealed 5'-PO 4 -70Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel B : Representative gel image showing digestion of the Blunt ended DNA substrate (annealed 5'-PO 4 -50Cy3 + 50blunt oligonucleotides) by SXT-Exo. Panel C : Representative gel image showing digestion of the 3'-overhang DNA substrate (annealed 5'-PO 4 -50Cy3 + 70overhang oligonucleotides) by SXT-Exo. Panel D : Plot showing the digestion of the three DNA substrates by SXT-Exo over a 20 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Labeling, Fluorescence, Incubation, Standard Deviation

    Processivity of SXT-Exo digestion of double stranded DNA . Heparin-trap experiments were used to calculate the average number of nucleotides hydrolyzed by an SXT-Exo trimer during a single binding event. SXT-Exo (41 nmol of trimers) and PstI-linearized pUC18 DNA (2686 bp in length; 18.1 pmol) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 s, before trapping unbound protein by addition of a large excess of heparin. Aliquots were removed at 0, 1, 2, 5, 10, 20 and 30 minutes; quenched, then dsDNA levels immediately quantified using PicoGreen assays to determine the number of nucleotides digested from each end (red circles) at each time point. Analogous control experiments without heparin were performed (black circles). Four independent replicates of each experiment were conducted, and graphs show the mean values ± standard deviation. See materials section for details.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Processivity of SXT-Exo digestion of double stranded DNA . Heparin-trap experiments were used to calculate the average number of nucleotides hydrolyzed by an SXT-Exo trimer during a single binding event. SXT-Exo (41 nmol of trimers) and PstI-linearized pUC18 DNA (2686 bp in length; 18.1 pmol) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 s, before trapping unbound protein by addition of a large excess of heparin. Aliquots were removed at 0, 1, 2, 5, 10, 20 and 30 minutes; quenched, then dsDNA levels immediately quantified using PicoGreen assays to determine the number of nucleotides digested from each end (red circles) at each time point. Analogous control experiments without heparin were performed (black circles). Four independent replicates of each experiment were conducted, and graphs show the mean values ± standard deviation. See materials section for details.

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Binding Assay, Incubation, Standard Deviation

    Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease . Panel A : Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl 2 ; λ-HindIII (NEB) DNA ladder (lane1). Panel B : Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl 2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C : Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl 2 ; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease . Panel A : Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl 2 ; λ-HindIII (NEB) DNA ladder (lane1). Panel B : Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl 2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C : Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl 2 ; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Agarose Gel Electrophoresis, Modification, Incubation

    Effects of addition of various monovalent and divalent salts on the double strand DNA activities of SXT-Exo, as determined by quenched PicoGreen assays . Panel A: Inhibition of the dsDNA exonuclease activities of SXT-Exo with sodium chloride (black squares), sodium phosphate (buffered to pH7.4; red circles) and sodium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (NaCl, Na 2 HPO 4 (pH7.4), or Na 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Panel B: Inhibition of the dsDNA exonuclease activities of SXT-Exo with potassium chloride (black squares), calcium chloride (red squares) and potassium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (KCl, CaCl 2 or K 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Relative dsDNA exonuclease activities were calculated (as a percentage) by comparison with results from analogous assays that contained: SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Effects of addition of various monovalent and divalent salts on the double strand DNA activities of SXT-Exo, as determined by quenched PicoGreen assays . Panel A: Inhibition of the dsDNA exonuclease activities of SXT-Exo with sodium chloride (black squares), sodium phosphate (buffered to pH7.4; red circles) and sodium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (NaCl, Na 2 HPO 4 (pH7.4), or Na 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Panel B: Inhibition of the dsDNA exonuclease activities of SXT-Exo with potassium chloride (black squares), calcium chloride (red squares) and potassium sulfate (blue triangles). SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 ; as well as the salt indicated in the figure (KCl, CaCl 2 or K 2 SO 4 ; 0-500 mM); were incubated at 37°C for 30 mins. Relative dsDNA exonuclease activities were calculated (as a percentage) by comparison with results from analogous assays that contained: SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) in Tris-HCl (25 mM, pH7.4), 0.5 mM MnCl 2 , 50 mM NaCl. Graphs show the the mean values ± standard deviation. See methods for detailed experimental procedures.

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Inhibition, Incubation, Standard Deviation

    Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins . Panel A . SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B . In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl 2 ; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P

    Journal: BMC Molecular Biology

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    doi: 10.1186/1471-2199-12-16

    Figure Lengend Snippet: Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins . Panel A . SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B . In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl 2 ; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P

    Article Snippet: All oligonucleotides, linear dsDNA and plasmid DNA were transformed into E. coli cells by electroporation using a MicroPulser electroporator with 1 mm gap electroporation cuvettes (BioRad).

    Techniques: Incubation, Standard Deviation

    Inhibition of R.PabI activities by methylation. ( A ) Inhibition of strand cleavage. Plasmid pBAD30_ cviQIM (Supplementary Figure S1) with a gene for M.CviQI, which generates 5′-GTm6AC as M.PabI does, under control of the pBAD promoter was prepared from cultures with varying concentrations of arabinose, its inducer. After incubation with R.PabI at 85°C for 6 h, the plasmid DNAs were subjected to 0.8% agarose gel electrophoresis. OC, open circle; SC, supercoiled; P, product DNA. Left lane: 1 kb DNA Ladder. ( B ) Strand-specific inhibition of cleavage in hemimethylated double-stranded DNA. A 40-mer single-stranded (GTAC40T or GTAC40Tme, Supplementary Table S2) or double-stranded (GTAC40_hemi_met or GTAC40, Supplementary Table S2) substrate (1 pmol, 100 nM) with a 32 P-label (black dot) at the 5′-end of either strand was incubated with R.PabI (9.2 pmol, 920 nM) at 85°C for 3 h. Products were separated by 10% denaturing PAGE. Box, recognition sequence (5′-GTAC). Me diamond, methylation of the top strand. Cleavage at the recognition sequence resulted in 27-mer and 13-mer oligonucleotides. The supershifted bands near the top of the gel are likely DNA–R.PabI complexes (see also Figure 5 and related text). ( C ) Inhibition of DNA glycosylase. A 40-mer double-stranded substrate (GTAC40 or GTAC40_full_met (Supplementary Table S2), 1 pmol, 100 nM) with a 32 P -label at the 5′ end of both strands was incubated with R.PabI (9.2 pmol, 920 nM) and then treated with 0.1 M DMED at 37°C for 1 h. Products were separated by 10% denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

    doi: 10.1093/nar/gkv116

    Figure Lengend Snippet: Inhibition of R.PabI activities by methylation. ( A ) Inhibition of strand cleavage. Plasmid pBAD30_ cviQIM (Supplementary Figure S1) with a gene for M.CviQI, which generates 5′-GTm6AC as M.PabI does, under control of the pBAD promoter was prepared from cultures with varying concentrations of arabinose, its inducer. After incubation with R.PabI at 85°C for 6 h, the plasmid DNAs were subjected to 0.8% agarose gel electrophoresis. OC, open circle; SC, supercoiled; P, product DNA. Left lane: 1 kb DNA Ladder. ( B ) Strand-specific inhibition of cleavage in hemimethylated double-stranded DNA. A 40-mer single-stranded (GTAC40T or GTAC40Tme, Supplementary Table S2) or double-stranded (GTAC40_hemi_met or GTAC40, Supplementary Table S2) substrate (1 pmol, 100 nM) with a 32 P-label (black dot) at the 5′-end of either strand was incubated with R.PabI (9.2 pmol, 920 nM) at 85°C for 3 h. Products were separated by 10% denaturing PAGE. Box, recognition sequence (5′-GTAC). Me diamond, methylation of the top strand. Cleavage at the recognition sequence resulted in 27-mer and 13-mer oligonucleotides. The supershifted bands near the top of the gel are likely DNA–R.PabI complexes (see also Figure 5 and related text). ( C ) Inhibition of DNA glycosylase. A 40-mer double-stranded substrate (GTAC40 or GTAC40_full_met (Supplementary Table S2), 1 pmol, 100 nM) with a 32 P -label at the 5′ end of both strands was incubated with R.PabI (9.2 pmol, 920 nM) and then treated with 0.1 M DMED at 37°C for 1 h. Products were separated by 10% denaturing PAGE.

    Article Snippet: After 0.5 h at 37°C, plasmid DNA was purified with a NucleoSpin Extract II (MACHEREY-NAGEL) and 0.11 pmol (200 ng) of plasmid DNA was mixed with a competent cell suspension with ∼108 bacteria (E. coli HST08 Premium Competent Cells, TaKaRa; Supplementary Table S1) thawed on ice.

    Techniques: Inhibition, Methylation, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Sequencing

    R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

    Journal: Nucleic Acids Research

    Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

    doi: 10.1093/nar/gkv116

    Figure Lengend Snippet: R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

    Article Snippet: After 0.5 h at 37°C, plasmid DNA was purified with a NucleoSpin Extract II (MACHEREY-NAGEL) and 0.11 pmol (200 ng) of plasmid DNA was mixed with a competent cell suspension with ∼108 bacteria (E. coli HST08 Premium Competent Cells, TaKaRa; Supplementary Table S1) thawed on ice.

    Techniques: Incubation, Concentration Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    R.PabI restricted DNA without strand breaks. ( A ) Design. Double-stranded, circular plasmid DNA was treated with R.PabI and transformation activity is measured. Box, recognition sequence. Me, base methylation within the recognition sequence. Only one recognition sequence is shown for simplicity. ( B ) R.PabI treatment at 37°C does not cause single-strand breaks. Plasmid pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) purified from E. coli under noninducing conditions was incubated with or without R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min, which was followed by proteinase K (ProK) treatment at 37°C overnight to inactivate R.PabI. DNA was incubated at 85°C for 6 h. Alternatively, plasmid was incubated with or without R.PabI at 85°C for 6 h. Samples were subjected to 0.8% agarose gel electrophoresis. OC, open circle; SC, supercoiled circle; P, product DNA. Left lane: 1 kb DNA ladder. ( C ) Loss of DNA transformation after R.PabI treatment. pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) with varying levels of methylation was treated with R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min and purified for quantitative transformation (‘Materials and Methods’ section, Supplementary Figure S5) of E. coli . The plasmid was prepared from cultures with 0.2% arabinose overnight, 0.2% arabinose 5 h, 0.02% arabinose 5 h, 0.002% arabinose 5 h, 0.0002% arabinose 5 h, and glucose overnight, in the order of expected decreased methylation level. E. coli HST08 was transformed with R.PabI-treated plasmids, and resulting colonies were counted to determine transformation efficiencies.

    Journal: Nucleic Acids Research

    Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

    doi: 10.1093/nar/gkv116

    Figure Lengend Snippet: R.PabI restricted DNA without strand breaks. ( A ) Design. Double-stranded, circular plasmid DNA was treated with R.PabI and transformation activity is measured. Box, recognition sequence. Me, base methylation within the recognition sequence. Only one recognition sequence is shown for simplicity. ( B ) R.PabI treatment at 37°C does not cause single-strand breaks. Plasmid pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) purified from E. coli under noninducing conditions was incubated with or without R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min, which was followed by proteinase K (ProK) treatment at 37°C overnight to inactivate R.PabI. DNA was incubated at 85°C for 6 h. Alternatively, plasmid was incubated with or without R.PabI at 85°C for 6 h. Samples were subjected to 0.8% agarose gel electrophoresis. OC, open circle; SC, supercoiled circle; P, product DNA. Left lane: 1 kb DNA ladder. ( C ) Loss of DNA transformation after R.PabI treatment. pBAD30_ cviQIM (0.11 pmol, 11 nM, Supplementary Figure S1) with varying levels of methylation was treated with R.PabI (0.77 pmol, 77 nM) at 37°C for 30 min and purified for quantitative transformation (‘Materials and Methods’ section, Supplementary Figure S5) of E. coli . The plasmid was prepared from cultures with 0.2% arabinose overnight, 0.2% arabinose 5 h, 0.02% arabinose 5 h, 0.002% arabinose 5 h, 0.0002% arabinose 5 h, and glucose overnight, in the order of expected decreased methylation level. E. coli HST08 was transformed with R.PabI-treated plasmids, and resulting colonies were counted to determine transformation efficiencies.

    Article Snippet: After 0.5 h at 37°C, plasmid DNA was purified with a NucleoSpin Extract II (MACHEREY-NAGEL) and 0.11 pmol (200 ng) of plasmid DNA was mixed with a competent cell suspension with ∼108 bacteria (E. coli HST08 Premium Competent Cells, TaKaRa; Supplementary Table S1) thawed on ice.

    Techniques: Plasmid Preparation, Transformation Assay, Activity Assay, Sequencing, Methylation, Purification, Incubation, Agarose Gel Electrophoresis

    The presence of exon 5 does not affect protein degradation but exhibits a profound impact on mA3 protein synthesis. (A) 293T cells were transfected with pFLAG-CMV2- mA3 d or pFLAG-CMV2- mA3 d Δ5 along with pFLAG-CMV2-GFP, which expresses green fluorescent protein (GFP) as a loading control. The cells were treated with cycloheximide for the indicated duration to stop protein synthesis and then harvested. DMSO was used as a solvent control. The FLAG-tagged mA3 and GFP were detected with the anti-FLAG antibody in immunoblotting. Endogenous IκBα expression was used as a positive control to confirm the effect of cycloheximide. (B) In vitro transcription and translation assays. DNA templates containing the entire coding region of the BALB/c full-length or Δ5 mA3 cDNA with the T7 promoter and His-tag sequence were subjected to an in vitro transcription/translation reaction by incubating for 30 or 60 min. The levels of mA3 protein synthesis and mRNA expression were evaluated by immunoblotting using the anti-His antibody and RT-PCR using the primer set a-b, respectively. Intensities of protein bands on the immunoblot membrane and DNA bands after the RT-PCR reaction and electrophoresis were measured by densitometry and are shown below each corresponding band. *, signals below detection limits.

    Journal: PLoS Pathogens

    Article Title: Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

    doi: 10.1371/journal.ppat.1002478

    Figure Lengend Snippet: The presence of exon 5 does not affect protein degradation but exhibits a profound impact on mA3 protein synthesis. (A) 293T cells were transfected with pFLAG-CMV2- mA3 d or pFLAG-CMV2- mA3 d Δ5 along with pFLAG-CMV2-GFP, which expresses green fluorescent protein (GFP) as a loading control. The cells were treated with cycloheximide for the indicated duration to stop protein synthesis and then harvested. DMSO was used as a solvent control. The FLAG-tagged mA3 and GFP were detected with the anti-FLAG antibody in immunoblotting. Endogenous IκBα expression was used as a positive control to confirm the effect of cycloheximide. (B) In vitro transcription and translation assays. DNA templates containing the entire coding region of the BALB/c full-length or Δ5 mA3 cDNA with the T7 promoter and His-tag sequence were subjected to an in vitro transcription/translation reaction by incubating for 30 or 60 min. The levels of mA3 protein synthesis and mRNA expression were evaluated by immunoblotting using the anti-His antibody and RT-PCR using the primer set a-b, respectively. Intensities of protein bands on the immunoblot membrane and DNA bands after the RT-PCR reaction and electrophoresis were measured by densitometry and are shown below each corresponding band. *, signals below detection limits.

    Article Snippet: Reciprocal chimeras between the above B5 and BALB exon 5–6 plasmids were generated by amplifying a linearized plasmid DNA lacking the 3′ intron 5 and exon 6 using primer pair G, and by amplifying the insert fragment using primer pair H. Site-directed mutagenesis was performed by employing the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the following templates and primers, listed separately in : BALB 100bp intron 5 (3′-100bp) plasmid was used as a common template for the preparation of BALB C14T, BALB C88G, and BALB C153GG163A with primer pairs I, J, K, respectively: B6 100bp intron 5 (3′-100bp) plasmid was used as a template to make B6 T14C or B6 G88C with primer pair L or M, respectively.

    Techniques: Transfection, Expressing, Positive Control, In Vitro, Sequencing, Reverse Transcription Polymerase Chain Reaction, Electrophoresis

    Protein expression of the full-length and Δ5 mA3 in transiently transfected 293T cells. (A) Exon 5-containing (5+) and Δ5 mA3 cDNA were tagged with the FLAG epitope and inserted into the expression vector. The arrows indicate the positions of the PCR primers. The primer set a-b is the same as shown in Figure 1 . (B and C) 293T cells were transfected with pFLAG-CMV2- mA3 d or pFLAG-CMV2- mA3 d Δ5 in B or with pFLAG-CMV2- mA3 b or pFLAG-CMV2- mA3 b Δ5 in C, which express either the 5+ or Δ5 mA3 cDNA cloned from BALB/c or B6 mice, respectively [27] . A luciferase-expressing plasmid, p luc , was co-transfected to standardize transfection efficiencies. At 24 hours after transfection, each one-third of the transfected cells was used for immunoblotting, RNA extraction, and luciferase assays. For immunoblotting, the full-length and Δ5 mA3 proteins were detected with the anti-FLAG antibody. Quantitative real-time PCR reactions were carried out with primer set a-b and were normalized with the levels of actin transcripts expressed in 293T cells. Data shown are averages of three reaction wells and SD. (D) RT-PCR assays were performed with (RT+) or without (RT−) reverse transcription to demonstrate specific detection of expected mRNA. Both primer sets e-f and a-b were used to detect mA3 mRNA. NC, negative control transfected with the empty vector, pFLAG-CMV2. RT– samples were included to evaluate the possible contamination of transfected DNA. Specific amplification of cDNA generated in the presence of RT was observed in the cells transfected with a plasmid expressing the 5+ or Δ5 mA3 cDNA. Similar results were obtained for the corresponding B6-derived cDNA clones.

    Journal: PLoS Pathogens

    Article Title: Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

    doi: 10.1371/journal.ppat.1002478

    Figure Lengend Snippet: Protein expression of the full-length and Δ5 mA3 in transiently transfected 293T cells. (A) Exon 5-containing (5+) and Δ5 mA3 cDNA were tagged with the FLAG epitope and inserted into the expression vector. The arrows indicate the positions of the PCR primers. The primer set a-b is the same as shown in Figure 1 . (B and C) 293T cells were transfected with pFLAG-CMV2- mA3 d or pFLAG-CMV2- mA3 d Δ5 in B or with pFLAG-CMV2- mA3 b or pFLAG-CMV2- mA3 b Δ5 in C, which express either the 5+ or Δ5 mA3 cDNA cloned from BALB/c or B6 mice, respectively [27] . A luciferase-expressing plasmid, p luc , was co-transfected to standardize transfection efficiencies. At 24 hours after transfection, each one-third of the transfected cells was used for immunoblotting, RNA extraction, and luciferase assays. For immunoblotting, the full-length and Δ5 mA3 proteins were detected with the anti-FLAG antibody. Quantitative real-time PCR reactions were carried out with primer set a-b and were normalized with the levels of actin transcripts expressed in 293T cells. Data shown are averages of three reaction wells and SD. (D) RT-PCR assays were performed with (RT+) or without (RT−) reverse transcription to demonstrate specific detection of expected mRNA. Both primer sets e-f and a-b were used to detect mA3 mRNA. NC, negative control transfected with the empty vector, pFLAG-CMV2. RT– samples were included to evaluate the possible contamination of transfected DNA. Specific amplification of cDNA generated in the presence of RT was observed in the cells transfected with a plasmid expressing the 5+ or Δ5 mA3 cDNA. Similar results were obtained for the corresponding B6-derived cDNA clones.

    Article Snippet: Reciprocal chimeras between the above B5 and BALB exon 5–6 plasmids were generated by amplifying a linearized plasmid DNA lacking the 3′ intron 5 and exon 6 using primer pair G, and by amplifying the insert fragment using primer pair H. Site-directed mutagenesis was performed by employing the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the following templates and primers, listed separately in : BALB 100bp intron 5 (3′-100bp) plasmid was used as a common template for the preparation of BALB C14T, BALB C88G, and BALB C153GG163A with primer pairs I, J, K, respectively: B6 100bp intron 5 (3′-100bp) plasmid was used as a template to make B6 T14C or B6 G88C with primer pair L or M, respectively.

    Techniques: Expressing, Transfection, FLAG-tag, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Mouse Assay, Luciferase, RNA Extraction, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Amplification, Generated, Derivative Assay

    The effect of exon 5 and its downstream sequences on mA3 intron 5 splicing. (A) Distributions of sequence polymorphisms in the intron 5 of the Apobec3 gene locus between the B6 allele [NT_039621] and the Celera database sequence of mixed mouse DNA [NW_001030577.1]. Shorter horizontal lines below the thick one representing intron 5 indicate regions of sequenced BALB/c genome [DDBJ accession No. AB646261-AB646265], which show nucleotide sequences identical to corresponding Celera database sequences. Spans of the analyzed regions are 1–620, 661–1597, 1643–2058, 2759–3443, and 5558–6247 in base numbers starting from the first nucleotide of intron 5. SNP are shown with vertical lines, single-base indels with arrows, and deletions of ≥2 bases with triangles. Indels and deletions above the thick horizontal line that represents intron 5 are deletions in the B6 allele relative to the Celera sequence, while those underneath the horizontal line are deletions in the Celera sequence relative to the B6 allele sequence. (B) Plasmid constructions for the splicing assays. The exon 5–6 plasmids harbored either the B6 or BALB/c genomic fragment encompassing exons 5 and 6, including the entire intron 5 of the corresponding allele. Each of the remaining plasmids possessed a sequentially reduced length of the intron 5 as indicated. The precise size of each PCR-generated 5′ fragment included was as follows: 3177bp and 3185bp for B6 and BALB/c intron 5-Δ3′; 2106bp and 2086bp for B6 and BALB/c 200bp intron 5; 1120bp and 1110bp for B6 and BALB/c 1100bp intron 5; and 634bp and 620bp for B6 and BALB/c 600bp intron 5, respectively. The primers g–h are the same as shown in Figure 4 . (C) RT-PCR detection of spliced messages expressed from the B6 and BALB/c exon 5–6 plasmids along with those from the exon 4–7 plasmids as controls. Primers g and h were used. A lack of expression of the spliced message in cells transfected with the B6 exon 5–6 plasmid was evident. (D and F) Splicing assays using intron 5 deletion plasmids. The intron 5 fragment included in each plasmid is shown in (B). RT-PCR assays were performed with primers g and h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies among the samples as shown in (C). Comparable levels of luciferase activities were observed in all samples in each experiment (data not shown). Quantitative real-time PCR data show averages of three reaction wells and SD. (E) Reciprocal chimeras were produced between B6 and BALB/c exon 5–6 by exchanging the cloned genomic DNA fragment at position 1100 within intron 5. The exact location of the above position 1100 for B6 and BALB/c intron 5 is described in the legend for (B). RT-PCR detection of spliced messages was performed with primers g and h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies.

    Journal: PLoS Pathogens

    Article Title: Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

    doi: 10.1371/journal.ppat.1002478

    Figure Lengend Snippet: The effect of exon 5 and its downstream sequences on mA3 intron 5 splicing. (A) Distributions of sequence polymorphisms in the intron 5 of the Apobec3 gene locus between the B6 allele [NT_039621] and the Celera database sequence of mixed mouse DNA [NW_001030577.1]. Shorter horizontal lines below the thick one representing intron 5 indicate regions of sequenced BALB/c genome [DDBJ accession No. AB646261-AB646265], which show nucleotide sequences identical to corresponding Celera database sequences. Spans of the analyzed regions are 1–620, 661–1597, 1643–2058, 2759–3443, and 5558–6247 in base numbers starting from the first nucleotide of intron 5. SNP are shown with vertical lines, single-base indels with arrows, and deletions of ≥2 bases with triangles. Indels and deletions above the thick horizontal line that represents intron 5 are deletions in the B6 allele relative to the Celera sequence, while those underneath the horizontal line are deletions in the Celera sequence relative to the B6 allele sequence. (B) Plasmid constructions for the splicing assays. The exon 5–6 plasmids harbored either the B6 or BALB/c genomic fragment encompassing exons 5 and 6, including the entire intron 5 of the corresponding allele. Each of the remaining plasmids possessed a sequentially reduced length of the intron 5 as indicated. The precise size of each PCR-generated 5′ fragment included was as follows: 3177bp and 3185bp for B6 and BALB/c intron 5-Δ3′; 2106bp and 2086bp for B6 and BALB/c 200bp intron 5; 1120bp and 1110bp for B6 and BALB/c 1100bp intron 5; and 634bp and 620bp for B6 and BALB/c 600bp intron 5, respectively. The primers g–h are the same as shown in Figure 4 . (C) RT-PCR detection of spliced messages expressed from the B6 and BALB/c exon 5–6 plasmids along with those from the exon 4–7 plasmids as controls. Primers g and h were used. A lack of expression of the spliced message in cells transfected with the B6 exon 5–6 plasmid was evident. (D and F) Splicing assays using intron 5 deletion plasmids. The intron 5 fragment included in each plasmid is shown in (B). RT-PCR assays were performed with primers g and h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies among the samples as shown in (C). Comparable levels of luciferase activities were observed in all samples in each experiment (data not shown). Quantitative real-time PCR data show averages of three reaction wells and SD. (E) Reciprocal chimeras were produced between B6 and BALB/c exon 5–6 by exchanging the cloned genomic DNA fragment at position 1100 within intron 5. The exact location of the above position 1100 for B6 and BALB/c intron 5 is described in the legend for (B). RT-PCR detection of spliced messages was performed with primers g and h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies.

    Article Snippet: Reciprocal chimeras between the above B5 and BALB exon 5–6 plasmids were generated by amplifying a linearized plasmid DNA lacking the 3′ intron 5 and exon 6 using primer pair G, and by amplifying the insert fragment using primer pair H. Site-directed mutagenesis was performed by employing the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the following templates and primers, listed separately in : BALB 100bp intron 5 (3′-100bp) plasmid was used as a common template for the preparation of BALB C14T, BALB C88G, and BALB C153GG163A with primer pairs I, J, K, respectively: B6 100bp intron 5 (3′-100bp) plasmid was used as a template to make B6 T14C or B6 G88C with primer pair L or M, respectively.

    Techniques: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Generated, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Real-time Polymerase Chain Reaction, Produced, Clone Assay

    The impact of TCCT repeat numbers on exon 5 inclusion in mA3 mRNA splicing. (A) Schematic representation of the genomic DNA structure of the Apobec3 gene locus. Boxes indicate exons. LTR, the endogenous retroviral LTR inserted into intron 2 in some strains of mice [37] . (B) Insert structures of the plasmids used for the splicing assays. BALB/c exon 4–7 and BALB/c ΔTCCT plasmids harbored the same DNA fragment amplified from BALB/c genomic DNA encompassing exons 4 and 7, except that the BALB/c ΔTCCT contains only a single TCCT quadruplet. B6 exon 4–7 plasmid harbored the B6 genomic DNA fragment encompassing exons 4 and 7. C741T and T741C indicate a C to T or reciprocal nucleotide substitution, respectively, within intron 4 at 741-bp downstream from the first nucleotide of exon 4 on the backbone of the BALB/c or B6 exon 4–7 insert. An additional repeat of the TCCT quadruplet was added to the B6 exon 4–6 construct to generate B6 +TCCT. The positions of primers g, h, i, and j used for RT-PCR assays are indicated with the arrows. (C) The plasmid harboring each insert depicted in (B) was transfected into BALB/3T3 cells, total RNA was extracted, and RT-PCR reactions were performed with primer pairs g–h and i–h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies among the samples. Comparable levels of luciferase activities were observed in all samples in each experiment (data not shown). The predicted splicing products are schematically indicated on the right side of the panel. Quantitative real-time PCR assays were performed with primers g and j, and data are shown here by averages of three reaction wells and SD.

    Journal: PLoS Pathogens

    Article Title: Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

    doi: 10.1371/journal.ppat.1002478

    Figure Lengend Snippet: The impact of TCCT repeat numbers on exon 5 inclusion in mA3 mRNA splicing. (A) Schematic representation of the genomic DNA structure of the Apobec3 gene locus. Boxes indicate exons. LTR, the endogenous retroviral LTR inserted into intron 2 in some strains of mice [37] . (B) Insert structures of the plasmids used for the splicing assays. BALB/c exon 4–7 and BALB/c ΔTCCT plasmids harbored the same DNA fragment amplified from BALB/c genomic DNA encompassing exons 4 and 7, except that the BALB/c ΔTCCT contains only a single TCCT quadruplet. B6 exon 4–7 plasmid harbored the B6 genomic DNA fragment encompassing exons 4 and 7. C741T and T741C indicate a C to T or reciprocal nucleotide substitution, respectively, within intron 4 at 741-bp downstream from the first nucleotide of exon 4 on the backbone of the BALB/c or B6 exon 4–7 insert. An additional repeat of the TCCT quadruplet was added to the B6 exon 4–6 construct to generate B6 +TCCT. The positions of primers g, h, i, and j used for RT-PCR assays are indicated with the arrows. (C) The plasmid harboring each insert depicted in (B) was transfected into BALB/3T3 cells, total RNA was extracted, and RT-PCR reactions were performed with primer pairs g–h and i–h. A portion of the transfected cells were utilized for luciferase assays to compare transfection efficiencies among the samples. Comparable levels of luciferase activities were observed in all samples in each experiment (data not shown). The predicted splicing products are schematically indicated on the right side of the panel. Quantitative real-time PCR assays were performed with primers g and j, and data are shown here by averages of three reaction wells and SD.

    Article Snippet: Reciprocal chimeras between the above B5 and BALB exon 5–6 plasmids were generated by amplifying a linearized plasmid DNA lacking the 3′ intron 5 and exon 6 using primer pair G, and by amplifying the insert fragment using primer pair H. Site-directed mutagenesis was performed by employing the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the following templates and primers, listed separately in : BALB 100bp intron 5 (3′-100bp) plasmid was used as a common template for the preparation of BALB C14T, BALB C88G, and BALB C153GG163A with primer pairs I, J, K, respectively: B6 100bp intron 5 (3′-100bp) plasmid was used as a template to make B6 T14C or B6 G88C with primer pair L or M, respectively.

    Techniques: Mouse Assay, Amplification, Plasmid Preparation, Construct, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Real-time Polymerase Chain Reaction

    Characterization of the late-promoter region of HPV18. (A) The strong activity of a P 811 promoter region is dependent on CCW orientation of the HPV18 Ori. HPV18-infected human foreskin keratinocytes (HFK18), differentiated by adding 2 mM calcium, were transfected for 48 h with each of the indicated plasmids. (A) Renilla luciferase plasmid pRL-TS was cotransfected as an internal control. The supernatant of the cell lysates from each transfection was examined for dual luciferase activities. Relative promoter activity levels were calculated by dividing the value representing the light unit readings from a testing promoter- firefly luciferase reporter by the value representing the light unit readings from the Renilla luciferase reporter (left panel). Plasmid pXHW61 directly derived from pXHW21 has the HPV18 Ori flipped into a CCW orientation (middle panel). Plasmid pXHW21-derived pXHW49 and pXHW22-derived pXHW50 have their corresponding promoter regions replaced by the SV40 early promoter derived from the pGL3 control vector (right panel). The data shown are means ± standard deviations (SD) of results from two to three independent experiments. P values were calculated using Student’s t test. (B) The P 811 promoter activity depends on keratinocyte differentiation. HFK18 cells with (+) or without (−) 2.0 mM calcium treatment were transfected with pXHW22 for 48 h and then analyzed for their luciferase activity. The data shown are from results from one of two experiments, with means ± SD calculated from triplicate samples. (C) Opposite orientations of the HPV18 Ori do not affect plasmid DNA replication in HFK18 cells. HPV18-infected HFK cells, differentiated by 2 mM calcium, were transfected with pXHW21 (Ori in CW orientation) or pXHW22 (Ori in CCW orientation) for 48 h. Replicated plasmid DNA isolated from the cells and the original input bacterial plasmid DNA were compared for their sensitivity to DpnI (digesting only methylated bacterial plasmid DNA) and MboI (digesting only unmethylated bacterial and replicated plasmid DNA). Because the input bacterial DNA is methylated at the adenine of GATC sequences, it is sensitive to digestion by DpnI but resistant to digestion by MboI (right panel), and because human cells lack adenine methylase activity, the newly replicated DNA lacking adenine methylation is thus resistant to DpnI digestion but susceptible to MboI digestion (left panel). ND, no digestion with a restriction enzyme. The digested DNA samples were then resolved in a 1% agarose gel and imaged by ethidium bromide staining. Lanes 1 and 8 represent DNA markers (M). (D) Aphidicolin, a DNA polymerase inhibitor, blocks CCW orientation-dependent HPV18 late promoter activity. The sensitivity of Ori-directed DNA replication and HPV18 late promoter activity in plasmid pXHW22 to aphidicolin at different doses was analyzed in the HFK18 cells cotransfected with plasmids pXHW22 and pRL-SV40 (an internal control). The supernatant of the cell lysates was examined for dual luciferase activities, and the relative promoter activity levels were calculated as described for panel A.

    Journal: mBio

    Article Title: Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

    doi: 10.1128/mBio.00713-17

    Figure Lengend Snippet: Characterization of the late-promoter region of HPV18. (A) The strong activity of a P 811 promoter region is dependent on CCW orientation of the HPV18 Ori. HPV18-infected human foreskin keratinocytes (HFK18), differentiated by adding 2 mM calcium, were transfected for 48 h with each of the indicated plasmids. (A) Renilla luciferase plasmid pRL-TS was cotransfected as an internal control. The supernatant of the cell lysates from each transfection was examined for dual luciferase activities. Relative promoter activity levels were calculated by dividing the value representing the light unit readings from a testing promoter- firefly luciferase reporter by the value representing the light unit readings from the Renilla luciferase reporter (left panel). Plasmid pXHW61 directly derived from pXHW21 has the HPV18 Ori flipped into a CCW orientation (middle panel). Plasmid pXHW21-derived pXHW49 and pXHW22-derived pXHW50 have their corresponding promoter regions replaced by the SV40 early promoter derived from the pGL3 control vector (right panel). The data shown are means ± standard deviations (SD) of results from two to three independent experiments. P values were calculated using Student’s t test. (B) The P 811 promoter activity depends on keratinocyte differentiation. HFK18 cells with (+) or without (−) 2.0 mM calcium treatment were transfected with pXHW22 for 48 h and then analyzed for their luciferase activity. The data shown are from results from one of two experiments, with means ± SD calculated from triplicate samples. (C) Opposite orientations of the HPV18 Ori do not affect plasmid DNA replication in HFK18 cells. HPV18-infected HFK cells, differentiated by 2 mM calcium, were transfected with pXHW21 (Ori in CW orientation) or pXHW22 (Ori in CCW orientation) for 48 h. Replicated plasmid DNA isolated from the cells and the original input bacterial plasmid DNA were compared for their sensitivity to DpnI (digesting only methylated bacterial plasmid DNA) and MboI (digesting only unmethylated bacterial and replicated plasmid DNA). Because the input bacterial DNA is methylated at the adenine of GATC sequences, it is sensitive to digestion by DpnI but resistant to digestion by MboI (right panel), and because human cells lack adenine methylase activity, the newly replicated DNA lacking adenine methylation is thus resistant to DpnI digestion but susceptible to MboI digestion (left panel). ND, no digestion with a restriction enzyme. The digested DNA samples were then resolved in a 1% agarose gel and imaged by ethidium bromide staining. Lanes 1 and 8 represent DNA markers (M). (D) Aphidicolin, a DNA polymerase inhibitor, blocks CCW orientation-dependent HPV18 late promoter activity. The sensitivity of Ori-directed DNA replication and HPV18 late promoter activity in plasmid pXHW22 to aphidicolin at different doses was analyzed in the HFK18 cells cotransfected with plasmids pXHW22 and pRL-SV40 (an internal control). The supernatant of the cell lysates was examined for dual luciferase activities, and the relative promoter activity levels were calculated as described for panel A.

    Article Snippet: DNA oligonucleotides oXHW391, oXHW392, oXHW393, and oXHW394 (70 nt in length) were cotransfected with plasmid DNA of pXHW15 or pXHW28 into HFK18 cells by electroporation using Amaxa 4-D-Nucleofector according to the protocol described by the manufacturer (Lonza).

    Techniques: Activity Assay, Infection, Transfection, Luciferase, Plasmid Preparation, Derivative Assay, Isolation, Methylation, Agarose Gel Electrophoresis, Staining

    Strand-biased effect on HPV18 Ori-directed DNA replication and HPV18 late promoter activity revealed by a single-stranded, 70-nt-long DNA oligonucleotide. (A) The maps of plasmids pXHW15 (left panel) and pXHW28 (right panel) and the relative positions and orientations (arrow directions) of paired oXHW391/oXHW392 and oXHW393/oXHW394 oligonucleotides. (B) Effects of individual oligonucleotides (oligos) on promoter activity in pXHW15- and pXHW28-transfected HFK18 cells. HFK18 cells were cotransfected with pXHW15 or pXHW28 at the indicated doses of individual oligonucleotides, along with plasmid pRL-TS, and were cultured in a complete culture medium supplemented with 2.0 mM calcium. Dual luciferase activities were analyzed and calculated at 48 h after transfection.

    Journal: mBio

    Article Title: Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

    doi: 10.1128/mBio.00713-17

    Figure Lengend Snippet: Strand-biased effect on HPV18 Ori-directed DNA replication and HPV18 late promoter activity revealed by a single-stranded, 70-nt-long DNA oligonucleotide. (A) The maps of plasmids pXHW15 (left panel) and pXHW28 (right panel) and the relative positions and orientations (arrow directions) of paired oXHW391/oXHW392 and oXHW393/oXHW394 oligonucleotides. (B) Effects of individual oligonucleotides (oligos) on promoter activity in pXHW15- and pXHW28-transfected HFK18 cells. HFK18 cells were cotransfected with pXHW15 or pXHW28 at the indicated doses of individual oligonucleotides, along with plasmid pRL-TS, and were cultured in a complete culture medium supplemented with 2.0 mM calcium. Dual luciferase activities were analyzed and calculated at 48 h after transfection.

    Article Snippet: DNA oligonucleotides oXHW391, oXHW392, oXHW393, and oXHW394 (70 nt in length) were cotransfected with plasmid DNA of pXHW15 or pXHW28 into HFK18 cells by electroporation using Amaxa 4-D-Nucleofector according to the protocol described by the manufacturer (Lonza).

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Cell Culture, Luciferase

    Identification of the cellular protein(s) bound to the repressor core element and its relation to HPV18 late promoter activity. (A) Expression of the repressor element-binding protein(s) in HFK18 cells depends on cell differentiation. 32 P-labeled, double-stranded DNA probe c and its c-4 mutant probe used for EMSA were incubated with NE of HFK18 cells cultured under low- or high-calcium conditions. HeLa NE served as a control. (B) The repressor element-binding protein(s) (~45 kDa) is detectable by Southwestern blotting. Biotin-labeled dsDNA oligonucleotide probe c (wt) and its c-4 mutant (mt) were used for protein pulldown assays. The proteins pulled down from HeLa NE were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, renatured, and probed by a 32 P-labeled double-stranded DNA oligonucleotide probe (c). Ctrl, control. (C) Isolation of the repressor-binding protein(s) for LC-MS/MS analysis. Biotinylated probe c (wt) or its c-4 mutant (mt) was immobilized on magnetic streptavidin beads and incubated with HeLa NE. After extensively washing, bound proteins on the beads were eluted with a buffer containing 0.5 M or 1.0 M NaCl. Eluted fractions and the eluted beads were analyzed by SDS-PAGE and visualized by Coomassie blue staining. The specific repressor element-binding proteins (indicated with arrows) corresponding to probe c were excised for LC-MS/MS analysis. M (lane 5), Bio-Rad broad-range protein markers (7, 14, 21, 31, 45, 66, 97, 116, and 200 kDa). (D) Verification of repressor element-binding proteins hnRNP D0B and hnRNP A/B from the bound proteins on the beads by Western blotting using corresponding antibodies. Pol, polymerase.

    Journal: mBio

    Article Title: Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

    doi: 10.1128/mBio.00713-17

    Figure Lengend Snippet: Identification of the cellular protein(s) bound to the repressor core element and its relation to HPV18 late promoter activity. (A) Expression of the repressor element-binding protein(s) in HFK18 cells depends on cell differentiation. 32 P-labeled, double-stranded DNA probe c and its c-4 mutant probe used for EMSA were incubated with NE of HFK18 cells cultured under low- or high-calcium conditions. HeLa NE served as a control. (B) The repressor element-binding protein(s) (~45 kDa) is detectable by Southwestern blotting. Biotin-labeled dsDNA oligonucleotide probe c (wt) and its c-4 mutant (mt) were used for protein pulldown assays. The proteins pulled down from HeLa NE were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, renatured, and probed by a 32 P-labeled double-stranded DNA oligonucleotide probe (c). Ctrl, control. (C) Isolation of the repressor-binding protein(s) for LC-MS/MS analysis. Biotinylated probe c (wt) or its c-4 mutant (mt) was immobilized on magnetic streptavidin beads and incubated with HeLa NE. After extensively washing, bound proteins on the beads were eluted with a buffer containing 0.5 M or 1.0 M NaCl. Eluted fractions and the eluted beads were analyzed by SDS-PAGE and visualized by Coomassie blue staining. The specific repressor element-binding proteins (indicated with arrows) corresponding to probe c were excised for LC-MS/MS analysis. M (lane 5), Bio-Rad broad-range protein markers (7, 14, 21, 31, 45, 66, 97, 116, and 200 kDa). (D) Verification of repressor element-binding proteins hnRNP D0B and hnRNP A/B from the bound proteins on the beads by Western blotting using corresponding antibodies. Pol, polymerase.

    Article Snippet: DNA oligonucleotides oXHW391, oXHW392, oXHW393, and oXHW394 (70 nt in length) were cotransfected with plasmid DNA of pXHW15 or pXHW28 into HFK18 cells by electroporation using Amaxa 4-D-Nucleofector according to the protocol described by the manufacturer (Lonza).

    Techniques: Activity Assay, Expressing, Binding Assay, Cell Differentiation, Labeling, Mutagenesis, Incubation, Cell Culture, Southwestern Blot, SDS Page, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining, Western Blot

    Identification of a transcriptional repressor element that affects P 811 promoter activity and binds cellular proteins. (A) Schematic diagrams and their promoter activities for individual plasmids derived from pXHW16 with indicated deletions of the promoter region (nt 417 to nt 850 in the HPV18 genome) inserted upstream of a firefly luciferase (Luc) gene. The numbers above the lines represent nucleotide positions of the first and last nucleotides of the insertion in the HPV18 genome. The HPV18 late TSS at nt position 811 is indicated by an arrow. HFK18 cells in the presence of 2 mM calcium were cotransfected for 48 h with the individual plasmids along with Renilla luciferase plasmid pRL-TS. The supernatant of the cell lysates was examined for dual luciferase activities, and the relative promoter activity levels were calculated as described for Fig. 2A . (B) Diagrams of the nucleotide positions of four synthetic, double-stranded DNA oligonucleotide probes (a to d) used for electrophoretic mobility shift assays (EMSA). (C) Probe c, derived from nt 573 to nt 598 in the HPV18 genome, interacts with a cellular protein(s) from HFK18 cells. Probes were labeled with 32 P, incubated with nuclear extract (NE) from HeLa or HFK18 cells, and then examined by EMSA. A Sp1 consensus oligonucleotide probe was used as a positive control. Protein-DNA complexes were resolved on a 4% native polyacrylamide gel. (D) Verification of the cellular proteins interacting with the repressor element by competitive gel shift assays. 32 P-labeled probe c and a 32 P-labeled Sp1 or TFIID (IID) oligonucleotide were incubated with NE prepared from HeLa or HFK18 cells in the presence or absence of an indicated cold competitor probe c, Sp1, TBP, or IID consensus oligonucleotide. Protein-DNA complexes were resolved on a 4% native polyacrylamide gel.

    Journal: mBio

    Article Title: Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

    doi: 10.1128/mBio.00713-17

    Figure Lengend Snippet: Identification of a transcriptional repressor element that affects P 811 promoter activity and binds cellular proteins. (A) Schematic diagrams and their promoter activities for individual plasmids derived from pXHW16 with indicated deletions of the promoter region (nt 417 to nt 850 in the HPV18 genome) inserted upstream of a firefly luciferase (Luc) gene. The numbers above the lines represent nucleotide positions of the first and last nucleotides of the insertion in the HPV18 genome. The HPV18 late TSS at nt position 811 is indicated by an arrow. HFK18 cells in the presence of 2 mM calcium were cotransfected for 48 h with the individual plasmids along with Renilla luciferase plasmid pRL-TS. The supernatant of the cell lysates was examined for dual luciferase activities, and the relative promoter activity levels were calculated as described for Fig. 2A . (B) Diagrams of the nucleotide positions of four synthetic, double-stranded DNA oligonucleotide probes (a to d) used for electrophoretic mobility shift assays (EMSA). (C) Probe c, derived from nt 573 to nt 598 in the HPV18 genome, interacts with a cellular protein(s) from HFK18 cells. Probes were labeled with 32 P, incubated with nuclear extract (NE) from HeLa or HFK18 cells, and then examined by EMSA. A Sp1 consensus oligonucleotide probe was used as a positive control. Protein-DNA complexes were resolved on a 4% native polyacrylamide gel. (D) Verification of the cellular proteins interacting with the repressor element by competitive gel shift assays. 32 P-labeled probe c and a 32 P-labeled Sp1 or TFIID (IID) oligonucleotide were incubated with NE prepared from HeLa or HFK18 cells in the presence or absence of an indicated cold competitor probe c, Sp1, TBP, or IID consensus oligonucleotide. Protein-DNA complexes were resolved on a 4% native polyacrylamide gel.

    Article Snippet: DNA oligonucleotides oXHW391, oXHW392, oXHW393, and oXHW394 (70 nt in length) were cotransfected with plasmid DNA of pXHW15 or pXHW28 into HFK18 cells by electroporation using Amaxa 4-D-Nucleofector according to the protocol described by the manufacturer (Lonza).

    Techniques: Activity Assay, Derivative Assay, Luciferase, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Positive Control

    Mapping of a protein-binding core motif from the HPV18 late-promoter repressor element by 3-bp linker-scanning mutational analysis in EMSA. (A) Sequence of probe c and its substitutions with a 3-bp GTT linker. (B) Identification of an AAGTATGCA motif in the repressor element as a protein-binding core. Indicated probes were 32 P-labeled double-stranded DNA oligonucleotide probe c and its derived variants in panel A used for EMSA with HeLa NE in the presence or absence of the indicated cold competitors. TBP and TFIID (IID) were included as controls. The protein-DNA complex was resolved on a 4% native polyacrylamide gel. (C) Protein binding profile of wild-type probe c and its c-4 mutant in EMSA. (D) Replacement of the probe c-corresponding sequence in plasmid pXHW16 with a c-4 sequence promotes HPV18 late promoter activity in HFK18 cells.

    Journal: mBio

    Article Title: Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

    doi: 10.1128/mBio.00713-17

    Figure Lengend Snippet: Mapping of a protein-binding core motif from the HPV18 late-promoter repressor element by 3-bp linker-scanning mutational analysis in EMSA. (A) Sequence of probe c and its substitutions with a 3-bp GTT linker. (B) Identification of an AAGTATGCA motif in the repressor element as a protein-binding core. Indicated probes were 32 P-labeled double-stranded DNA oligonucleotide probe c and its derived variants in panel A used for EMSA with HeLa NE in the presence or absence of the indicated cold competitors. TBP and TFIID (IID) were included as controls. The protein-DNA complex was resolved on a 4% native polyacrylamide gel. (C) Protein binding profile of wild-type probe c and its c-4 mutant in EMSA. (D) Replacement of the probe c-corresponding sequence in plasmid pXHW16 with a c-4 sequence promotes HPV18 late promoter activity in HFK18 cells.

    Article Snippet: DNA oligonucleotides oXHW391, oXHW392, oXHW393, and oXHW394 (70 nt in length) were cotransfected with plasmid DNA of pXHW15 or pXHW28 into HFK18 cells by electroporation using Amaxa 4-D-Nucleofector according to the protocol described by the manufacturer (Lonza).

    Techniques: Protein Binding, Sequencing, Labeling, Derivative Assay, Mutagenesis, Plasmid Preparation, Activity Assay

    Transfection efficiency of polycation lipid nanocarrier/DNA complexes (PDC) (N/P = 10) in human lung adenocarcinoma (SPC-A1) cells compared with those of Lipofectamine™ 2000/DNA complexes (LDC). ( A ) Transfection efficiency of plasmid pEGFP-N2 determined by flow cytometer; ( B ) relative luciferase activity in SPC-A1 cells treated with PDC in comparison with that of LDC; ( C ) the cell viability of PDCs in SPC-A1 cells compared with that of polyethylenimine (PEI)/DNA complexes at various N/P ratios (* P

    Journal: International Journal of Nanomedicine

    Article Title: Triolein-based polycation lipid nanocarrier for efficient gene delivery: characteristics and mechanism

    doi: 10.2147/IJN.S24720

    Figure Lengend Snippet: Transfection efficiency of polycation lipid nanocarrier/DNA complexes (PDC) (N/P = 10) in human lung adenocarcinoma (SPC-A1) cells compared with those of Lipofectamine™ 2000/DNA complexes (LDC). ( A ) Transfection efficiency of plasmid pEGFP-N2 determined by flow cytometer; ( B ) relative luciferase activity in SPC-A1 cells treated with PDC in comparison with that of LDC; ( C ) the cell viability of PDCs in SPC-A1 cells compared with that of polyethylenimine (PEI)/DNA complexes at various N/P ratios (* P

    Article Snippet: Laser confocal scanning microscopy To visualize the localization of PDC in SPC-A1 cells, plasmid DNA was fluorescently labeled with Label IT® TM-Rhodamine Labeling Kit (Mirus) according to the manufacturer’s protocol, and the fluorescently labeled PDC were prepared as described previously.

    Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Luciferase, Activity Assay

    Colocalization of polycation lipid nanocarrier/DNA complexes (PDC) (N/P = 10) with nucleus in human lung adenocarcinoma (SPC-A1) cells under laser scanning confocal microscopy at 6 hours after transfection. ( A ) TM-Rhodamine labeled plasmid DNA (red); ( B ) Hoechst 33258 labe led nuclei (blue); ( C ) phase-contrast image of SPC-A1 cells; ( D ) merged image, scale bar = 25 μm.

    Journal: International Journal of Nanomedicine

    Article Title: Triolein-based polycation lipid nanocarrier for efficient gene delivery: characteristics and mechanism

    doi: 10.2147/IJN.S24720

    Figure Lengend Snippet: Colocalization of polycation lipid nanocarrier/DNA complexes (PDC) (N/P = 10) with nucleus in human lung adenocarcinoma (SPC-A1) cells under laser scanning confocal microscopy at 6 hours after transfection. ( A ) TM-Rhodamine labeled plasmid DNA (red); ( B ) Hoechst 33258 labe led nuclei (blue); ( C ) phase-contrast image of SPC-A1 cells; ( D ) merged image, scale bar = 25 μm.

    Article Snippet: Laser confocal scanning microscopy To visualize the localization of PDC in SPC-A1 cells, plasmid DNA was fluorescently labeled with Label IT® TM-Rhodamine Labeling Kit (Mirus) according to the manufacturer’s protocol, and the fluorescently labeled PDC were prepared as described previously.

    Techniques: Confocal Microscopy, Transfection, Labeling, Plasmid Preparation

    Characteristics of polycation lipid nanocarrier (PLN) and PLN/DNA complexes (PDC). ( A ) Scheme of PLN composed of cetylated polyethylenimine (PEI) 1200 (c-PEI), dioleoyl phosphatidylethanolamine (DOPE), and triolein; ( B ) size distribution of PLN; ( C ) atomic force microscopy image of PLN, scale bar = 1.0 μm; ( D ) size distribution of PDC (N/P ratio = 10); ( E ) atomic force microscopy image of the PDC (N/P ratio = 10), scale bar = 1.0 μm; ( F ) agarose gel electrophoresis of the complexes of PLN with plasmid DNA stained with ethidium bromide.

    Journal: International Journal of Nanomedicine

    Article Title: Triolein-based polycation lipid nanocarrier for efficient gene delivery: characteristics and mechanism

    doi: 10.2147/IJN.S24720

    Figure Lengend Snippet: Characteristics of polycation lipid nanocarrier (PLN) and PLN/DNA complexes (PDC). ( A ) Scheme of PLN composed of cetylated polyethylenimine (PEI) 1200 (c-PEI), dioleoyl phosphatidylethanolamine (DOPE), and triolein; ( B ) size distribution of PLN; ( C ) atomic force microscopy image of PLN, scale bar = 1.0 μm; ( D ) size distribution of PDC (N/P ratio = 10); ( E ) atomic force microscopy image of the PDC (N/P ratio = 10), scale bar = 1.0 μm; ( F ) agarose gel electrophoresis of the complexes of PLN with plasmid DNA stained with ethidium bromide.

    Article Snippet: Laser confocal scanning microscopy To visualize the localization of PDC in SPC-A1 cells, plasmid DNA was fluorescently labeled with Label IT® TM-Rhodamine Labeling Kit (Mirus) according to the manufacturer’s protocol, and the fluorescently labeled PDC were prepared as described previously.

    Techniques: Microscopy, Agarose Gel Electrophoresis, Plasmid Preparation, Staining

    Transfection of pEGFP-N2 in human lung adenocarcinoma (SPC-A1) cells mediated with Lipofectamine™ 2000/DNA complexes (LDC) and polycation lipid nanocarrier/DNA complexes (PDC) (N/P = 10). ( A ) Images of SPC-A1 cells transfected by LDC, scale bar = 100 μm; ( B ) images of SPC-A1 cells mediated by PDC (N/P = 10), scale bar = 100 μm; ( C ) fluorescence intensity of expressed green fluorescent protein in SPC-A1 cells mediated by different PDCs with various N/P ratios (** P

    Journal: International Journal of Nanomedicine

    Article Title: Triolein-based polycation lipid nanocarrier for efficient gene delivery: characteristics and mechanism

    doi: 10.2147/IJN.S24720

    Figure Lengend Snippet: Transfection of pEGFP-N2 in human lung adenocarcinoma (SPC-A1) cells mediated with Lipofectamine™ 2000/DNA complexes (LDC) and polycation lipid nanocarrier/DNA complexes (PDC) (N/P = 10). ( A ) Images of SPC-A1 cells transfected by LDC, scale bar = 100 μm; ( B ) images of SPC-A1 cells mediated by PDC (N/P = 10), scale bar = 100 μm; ( C ) fluorescence intensity of expressed green fluorescent protein in SPC-A1 cells mediated by different PDCs with various N/P ratios (** P

    Article Snippet: Laser confocal scanning microscopy To visualize the localization of PDC in SPC-A1 cells, plasmid DNA was fluorescently labeled with Label IT® TM-Rhodamine Labeling Kit (Mirus) according to the manufacturer’s protocol, and the fluorescently labeled PDC were prepared as described previously.

    Techniques: Transfection, Fluorescence

    Liver distribution of pMMP13 in PEI or HA/PEI complexes . Mice were intravenously injected with pMMP13 (1 mg/kg) in PEI complexes or HA-shielded PEI complexes. At 4 hours after injection, gDNA was extracted from blood and liver tissues. pMMP13 copy numbers in the liver were divided by those in the blood. The data are the mean ± SE ( n = 5). gDNA, genomic DNA; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Journal: Molecular Therapy

    Article Title: Antifibrotic Effect of MMP13-encoding Plasmid DNA Delivered Using Polyethylenimine Shielded With Hyaluronic Acid

    doi: 10.1038/mt.2010.262

    Figure Lengend Snippet: Liver distribution of pMMP13 in PEI or HA/PEI complexes . Mice were intravenously injected with pMMP13 (1 mg/kg) in PEI complexes or HA-shielded PEI complexes. At 4 hours after injection, gDNA was extracted from blood and liver tissues. pMMP13 copy numbers in the liver were divided by those in the blood. The data are the mean ± SE ( n = 5). gDNA, genomic DNA; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Article Snippet: After treating for 48 hours with HA/PEI/pMMP13 ternary or PEI/pMMP13 binary complexes containing 2 µg of plasmid DNA, 20 µl of MTT (5 mg/ml) was added and cells were incubated for 3 hours.

    Techniques: Mouse Assay, Injection, Plasmid Preparation

    Survival rates of mice after intravenous administration of pMMP13 in PEI or HA/PEI complexes . Mice in each group ( n = 5) were intravenously injected with pMMP13 in PEI or HA-shielded PEI complexes. The behavior changes of survived mice were checked for 1-month postdose. HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Journal: Molecular Therapy

    Article Title: Antifibrotic Effect of MMP13-encoding Plasmid DNA Delivered Using Polyethylenimine Shielded With Hyaluronic Acid

    doi: 10.1038/mt.2010.262

    Figure Lengend Snippet: Survival rates of mice after intravenous administration of pMMP13 in PEI or HA/PEI complexes . Mice in each group ( n = 5) were intravenously injected with pMMP13 in PEI or HA-shielded PEI complexes. The behavior changes of survived mice were checked for 1-month postdose. HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Article Snippet: After treating for 48 hours with HA/PEI/pMMP13 ternary or PEI/pMMP13 binary complexes containing 2 µg of plasmid DNA, 20 µl of MTT (5 mg/ml) was added and cells were incubated for 3 hours.

    Techniques: Mouse Assay, Injection, Plasmid Preparation

    Expression of MMP13 proteins in liver tissues of fibrotic mice . Expression of EGFP and MMP13 proteins in liver tissues was visualized by fluorescent microscopy and immunoblotting, respectively. ( a , c ) Representative phase-contrast and ( b , d ) fluorescence images were shown for tissues from ( a , b ) untreated liver or ( c , d ) pMMP13-treated liver. Bar = 100 µm. The expression of MMP13 proteins in liver tissues was analyzed by ( e ) immunoblotting. EGFP, enriched green fluorescent protein; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Journal: Molecular Therapy

    Article Title: Antifibrotic Effect of MMP13-encoding Plasmid DNA Delivered Using Polyethylenimine Shielded With Hyaluronic Acid

    doi: 10.1038/mt.2010.262

    Figure Lengend Snippet: Expression of MMP13 proteins in liver tissues of fibrotic mice . Expression of EGFP and MMP13 proteins in liver tissues was visualized by fluorescent microscopy and immunoblotting, respectively. ( a , c ) Representative phase-contrast and ( b , d ) fluorescence images were shown for tissues from ( a , b ) untreated liver or ( c , d ) pMMP13-treated liver. Bar = 100 µm. The expression of MMP13 proteins in liver tissues was analyzed by ( e ) immunoblotting. EGFP, enriched green fluorescent protein; HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Article Snippet: After treating for 48 hours with HA/PEI/pMMP13 ternary or PEI/pMMP13 binary complexes containing 2 µg of plasmid DNA, 20 µl of MTT (5 mg/ml) was added and cells were incubated for 3 hours.

    Techniques: Expressing, Mouse Assay, Microscopy, Fluorescence, Plasmid Preparation

    Stability of pMMP13 in vitro and in vivo . ( a ) Naked pMMP13, PEI/pMMP13, or HA/PEI/pMMP13 was incubated with DNase I. The samples were collected at various time points. pMMP13 was extracted from the samples and loaded onto a 1% agarose gel. ( b ) Mice were injected intravenously with pMMP13 at 1 mg/kg dose in naked form or complexes. At 4 hours after injection, DNA was isolated from blood and pMMP13 copy numbers were determined by quantitative RT-PCR. The data are the mean ± SE ( n = 5). *Significantly higher than naked pMMP13 ( P

    Journal: Molecular Therapy

    Article Title: Antifibrotic Effect of MMP13-encoding Plasmid DNA Delivered Using Polyethylenimine Shielded With Hyaluronic Acid

    doi: 10.1038/mt.2010.262

    Figure Lengend Snippet: Stability of pMMP13 in vitro and in vivo . ( a ) Naked pMMP13, PEI/pMMP13, or HA/PEI/pMMP13 was incubated with DNase I. The samples were collected at various time points. pMMP13 was extracted from the samples and loaded onto a 1% agarose gel. ( b ) Mice were injected intravenously with pMMP13 at 1 mg/kg dose in naked form or complexes. At 4 hours after injection, DNA was isolated from blood and pMMP13 copy numbers were determined by quantitative RT-PCR. The data are the mean ± SE ( n = 5). *Significantly higher than naked pMMP13 ( P

    Article Snippet: After treating for 48 hours with HA/PEI/pMMP13 ternary or PEI/pMMP13 binary complexes containing 2 µg of plasmid DNA, 20 µl of MTT (5 mg/ml) was added and cells were incubated for 3 hours.

    Techniques: In Vitro, In Vivo, Incubation, Agarose Gel Electrophoresis, Mouse Assay, Injection, Isolation, Quantitative RT-PCR

    Schematic representation for the construction of pMMP13 and formation of HA-shielded PEI and plasmid DNA ternary complex for gene delivery . ( a ) MMP13 cDNA was inserted into the Bgl II/ Sal I sites of the pIRES2-EGFP expression vector encoding EGFP. The recombinant plasmid DNA encoding both MMP13 and EGFP was abbreviated as pMMP13. ( b ) Plasmid DNA was electrostatically complexed to cationic PEI, resulting in binary complexes. The binary complexes with surplus cationic charges were then coated with negatively charged HA, providing ternary complexes. cDNA, complementary DNA; EGFP, enriched green fluorescent protein; HA, hyaluronic acid; IRES, internal ribosome entry site; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Journal: Molecular Therapy

    Article Title: Antifibrotic Effect of MMP13-encoding Plasmid DNA Delivered Using Polyethylenimine Shielded With Hyaluronic Acid

    doi: 10.1038/mt.2010.262

    Figure Lengend Snippet: Schematic representation for the construction of pMMP13 and formation of HA-shielded PEI and plasmid DNA ternary complex for gene delivery . ( a ) MMP13 cDNA was inserted into the Bgl II/ Sal I sites of the pIRES2-EGFP expression vector encoding EGFP. The recombinant plasmid DNA encoding both MMP13 and EGFP was abbreviated as pMMP13. ( b ) Plasmid DNA was electrostatically complexed to cationic PEI, resulting in binary complexes. The binary complexes with surplus cationic charges were then coated with negatively charged HA, providing ternary complexes. cDNA, complementary DNA; EGFP, enriched green fluorescent protein; HA, hyaluronic acid; IRES, internal ribosome entry site; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Article Snippet: After treating for 48 hours with HA/PEI/pMMP13 ternary or PEI/pMMP13 binary complexes containing 2 µg of plasmid DNA, 20 µl of MTT (5 mg/ml) was added and cells were incubated for 3 hours.

    Techniques: Plasmid Preparation, Expressing, Recombinant

    Levels of MMP13 mRNA in liver tissues of fibrotic mice . ( a ) For induction of fibrosis, mice were intraperitoneally injected with CCl 4 and intravenously treated with HA/PEI/pVector or HA/PEI/pMMP13 according to the dosing schedule. The mRNA levels of MMP13 were measured from total RNA isolated from the liver tissue from normal mice, untreated fibrotic mice, or fibrotic mice treated with HA/PEI/pVector or HA/PEI/pMMP13. ( b ) The expression levels of MMP13 mRNA were normalized to those of GAPDH. The data are the mean ± SE ( n = 4). HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Journal: Molecular Therapy

    Article Title: Antifibrotic Effect of MMP13-encoding Plasmid DNA Delivered Using Polyethylenimine Shielded With Hyaluronic Acid

    doi: 10.1038/mt.2010.262

    Figure Lengend Snippet: Levels of MMP13 mRNA in liver tissues of fibrotic mice . ( a ) For induction of fibrosis, mice were intraperitoneally injected with CCl 4 and intravenously treated with HA/PEI/pVector or HA/PEI/pMMP13 according to the dosing schedule. The mRNA levels of MMP13 were measured from total RNA isolated from the liver tissue from normal mice, untreated fibrotic mice, or fibrotic mice treated with HA/PEI/pVector or HA/PEI/pMMP13. ( b ) The expression levels of MMP13 mRNA were normalized to those of GAPDH. The data are the mean ± SE ( n = 4). HA, hyaluronic acid; PEI, polyethylenimine; pMMP13, plasmid DNA encoding matrix metalloproteinases 13.

    Article Snippet: After treating for 48 hours with HA/PEI/pMMP13 ternary or PEI/pMMP13 binary complexes containing 2 µg of plasmid DNA, 20 µl of MTT (5 mg/ml) was added and cells were incubated for 3 hours.

    Techniques: Mouse Assay, Injection, Isolation, Expressing, Plasmid Preparation

    Generation of ΔN and ΔNC mice Diagrammatic representation of ΔN ( a ) and ΔNC ( b ) knock-in mouse line generation. The endogenous Mecp2 allele was targeted in male ES cells. The site of Cas9 cleavage in the WT sequence is shown by the scissors symbol (used for production of ΔN knock-in ES cells). The selection cassette was removed in vivo by crossing chimaeras with deleter ( CMV-Cre ) transgenic mice. Southern blot analysis shows correct targeting of ES cells and successful cassette deletion in the knock-in mice. The solid black line represents the sequence encoded in the targeted vector and the dotted lines indicate the flanking regions of mouse genomic DNA. For gel source data, see Supplementary Information .

    Journal: Nature

    Article Title: Radically truncated MeCP2 rescues Rett syndrome-like neurological defects

    doi: 10.1038/nature24058

    Figure Lengend Snippet: Generation of ΔN and ΔNC mice Diagrammatic representation of ΔN ( a ) and ΔNC ( b ) knock-in mouse line generation. The endogenous Mecp2 allele was targeted in male ES cells. The site of Cas9 cleavage in the WT sequence is shown by the scissors symbol (used for production of ΔN knock-in ES cells). The selection cassette was removed in vivo by crossing chimaeras with deleter ( CMV-Cre ) transgenic mice. Southern blot analysis shows correct targeting of ES cells and successful cassette deletion in the knock-in mice. The solid black line represents the sequence encoded in the targeted vector and the dotted lines indicate the flanking regions of mouse genomic DNA. For gel source data, see Supplementary Information .

    Article Snippet: MeCP2 localisation and TBL1X-mCherry recruitment assay NIH-3T3 cells were seeded on coverslips in 6-well plates (25,000 cells per well) and transfected with 2 μg plasmid DNA (pEGFPN1-MeCP2 alone or pEGFPN1-MeCP2 and pmCherry-TBL1X ) using JetPEI (PolyPlus Transfection).

    Techniques: Mouse Assay, Knock-In, Sequencing, Selection, In Vivo, Transgenic Assay, Southern Blot, Plasmid Preparation

    Generation of ΔNIC and STOP mice Diagrammatic representation of ΔNIC and STOP mouse line generation. The endogenous Mecp2 allele was targeted in male ES cells. The site of Cas9 cleavage in the WT sequence is shown by the scissors symbol. The selection cassette was removed in vivo by crossing chimaeras with deleter ( CMV-Cre ) transgenic mice to produce constitutively expressing ΔNIC mice, or retained to produce STOP mice. Southern blot analysis shows correct targeting of ES cells and successful cassette deletion in the ΔNIC knock-in mice. The solid black line represents the sequence encoded in the targeted vector and the dotted lines indicate the flanking regions of mouse genomic DNA. For gel source data, see Supplementary Information .

    Journal: Nature

    Article Title: Radically truncated MeCP2 rescues Rett syndrome-like neurological defects

    doi: 10.1038/nature24058

    Figure Lengend Snippet: Generation of ΔNIC and STOP mice Diagrammatic representation of ΔNIC and STOP mouse line generation. The endogenous Mecp2 allele was targeted in male ES cells. The site of Cas9 cleavage in the WT sequence is shown by the scissors symbol. The selection cassette was removed in vivo by crossing chimaeras with deleter ( CMV-Cre ) transgenic mice to produce constitutively expressing ΔNIC mice, or retained to produce STOP mice. Southern blot analysis shows correct targeting of ES cells and successful cassette deletion in the ΔNIC knock-in mice. The solid black line represents the sequence encoded in the targeted vector and the dotted lines indicate the flanking regions of mouse genomic DNA. For gel source data, see Supplementary Information .

    Article Snippet: MeCP2 localisation and TBL1X-mCherry recruitment assay NIH-3T3 cells were seeded on coverslips in 6-well plates (25,000 cells per well) and transfected with 2 μg plasmid DNA (pEGFPN1-MeCP2 alone or pEGFPN1-MeCP2 and pmCherry-TBL1X ) using JetPEI (PolyPlus Transfection).

    Techniques: Mouse Assay, Sequencing, Selection, In Vivo, Transgenic Assay, Expressing, Southern Blot, Knock-In, Plasmid Preparation

    Truncated MeCP2 proteins retain the ability to bind methylated DNA and the NCoR/SMRT complex a, EGFP-tagged truncated proteins immunoprecipitate components of the NCoR/SMRT co-repressor complex: NCoR, HDAC3 and TBL1XR1. WT and R306C were used as positive and negative controls for binding, respectively. ‘In’ = input, ‘IP’ = immunoprecipiate. For gel source data, see Supplementary Information . b, EGFP-tagged truncated MeCP2 proteins localise to mCpG-rich heterochromatic foci when overexpressed in mouse fibroblasts (NIH-3T3 cells). WT and R111G were used as controls to show focal and diffuse localisation, respectively. Scale bars indicate 10 µm. c, EGFP-tagged truncated proteins recruit TBL1X-mCherry to heterochromatin when co-overexpressed in NIH-3T3 cells. WT and R306C were used as positive and negative controls for TBL1X-mCherry recruitment, respectively. scale bars indicate 10 µm. Quantification (right) shows the percentage of cells with focal TBL1X-mCherry localisation, evaluated relative to WT using Fisher’s exact tests: R306C **** P

    Journal: Nature

    Article Title: Radically truncated MeCP2 rescues Rett syndrome-like neurological defects

    doi: 10.1038/nature24058

    Figure Lengend Snippet: Truncated MeCP2 proteins retain the ability to bind methylated DNA and the NCoR/SMRT complex a, EGFP-tagged truncated proteins immunoprecipitate components of the NCoR/SMRT co-repressor complex: NCoR, HDAC3 and TBL1XR1. WT and R306C were used as positive and negative controls for binding, respectively. ‘In’ = input, ‘IP’ = immunoprecipiate. For gel source data, see Supplementary Information . b, EGFP-tagged truncated MeCP2 proteins localise to mCpG-rich heterochromatic foci when overexpressed in mouse fibroblasts (NIH-3T3 cells). WT and R111G were used as controls to show focal and diffuse localisation, respectively. Scale bars indicate 10 µm. c, EGFP-tagged truncated proteins recruit TBL1X-mCherry to heterochromatin when co-overexpressed in NIH-3T3 cells. WT and R306C were used as positive and negative controls for TBL1X-mCherry recruitment, respectively. scale bars indicate 10 µm. Quantification (right) shows the percentage of cells with focal TBL1X-mCherry localisation, evaluated relative to WT using Fisher’s exact tests: R306C **** P

    Article Snippet: MeCP2 localisation and TBL1X-mCherry recruitment assay NIH-3T3 cells were seeded on coverslips in 6-well plates (25,000 cells per well) and transfected with 2 μg plasmid DNA (pEGFPN1-MeCP2 alone or pEGFPN1-MeCP2 and pmCherry-TBL1X ) using JetPEI (PolyPlus Transfection).

    Techniques: Methylation, Binding Assay

    Activation or viral transduction of ΔNIC ameliorates neurological phenotypes in MeCP2-deficient mice a, Timeline of Cre-mediated activation of ΔNIC induced by Tamoxifen injections. b, Phenotypic severity scores (mean ± SEM) of mice injected with Tamoxifen (arrows) from 4-28 weeks: WT ( n =4), WT CreER T ( n =4), STOP ( n =9) and STOP CreER ( n =9). c, Kaplan-Meier plot showing survival of the cohort shown in panel b . d, Diagram of the DNA sequence inserted into an scAAV viral vector, comprising a 426 nt Mecp2 promoter driving the human ΔNIC coding sequence plus a C-terminal Myc tag and 3’ UTR. A vector containing full-length human MECP2 25 is shown for comparison. e, Timeline of the scAAV-mediated gene therapy experiment. f, Phenotypic severity scores (mean ± SEM) of scAAV-injected mice from 5-30 weeks: WT + vehicle ( n =15), Mecp2- null + vehicle ( n =20) and Mecp2- null + hΔNIC ( n =17). g, Kaplan-Meier plot showing survival of the cohort shown in panel f . Four Mecp2 -null + hΔNIC animals reached their humane end-point. Five Mecp2 -null + ΔNIC animals were culled due to injuries unrelated to RTT-like phenotypes at 16, 23, 25, 26 and 29 weeks of age (data shown as ticks). Survival of Mecp2 -null + ΔNIC animals was compared to Mecp2 -null + vehicle controls using the Mantel-Cox test: P =

    Journal: Nature

    Article Title: Radically truncated MeCP2 rescues Rett syndrome-like neurological defects

    doi: 10.1038/nature24058

    Figure Lengend Snippet: Activation or viral transduction of ΔNIC ameliorates neurological phenotypes in MeCP2-deficient mice a, Timeline of Cre-mediated activation of ΔNIC induced by Tamoxifen injections. b, Phenotypic severity scores (mean ± SEM) of mice injected with Tamoxifen (arrows) from 4-28 weeks: WT ( n =4), WT CreER T ( n =4), STOP ( n =9) and STOP CreER ( n =9). c, Kaplan-Meier plot showing survival of the cohort shown in panel b . d, Diagram of the DNA sequence inserted into an scAAV viral vector, comprising a 426 nt Mecp2 promoter driving the human ΔNIC coding sequence plus a C-terminal Myc tag and 3’ UTR. A vector containing full-length human MECP2 25 is shown for comparison. e, Timeline of the scAAV-mediated gene therapy experiment. f, Phenotypic severity scores (mean ± SEM) of scAAV-injected mice from 5-30 weeks: WT + vehicle ( n =15), Mecp2- null + vehicle ( n =20) and Mecp2- null + hΔNIC ( n =17). g, Kaplan-Meier plot showing survival of the cohort shown in panel f . Four Mecp2 -null + hΔNIC animals reached their humane end-point. Five Mecp2 -null + ΔNIC animals were culled due to injuries unrelated to RTT-like phenotypes at 16, 23, 25, 26 and 29 weeks of age (data shown as ticks). Survival of Mecp2 -null + ΔNIC animals was compared to Mecp2 -null + vehicle controls using the Mantel-Cox test: P =

    Article Snippet: MeCP2 localisation and TBL1X-mCherry recruitment assay NIH-3T3 cells were seeded on coverslips in 6-well plates (25,000 cells per well) and transfected with 2 μg plasmid DNA (pEGFPN1-MeCP2 alone or pEGFPN1-MeCP2 and pmCherry-TBL1X ) using JetPEI (PolyPlus Transfection).

    Techniques: Activation Assay, Transduction, Mouse Assay, Injection, Sequencing, Plasmid Preparation

    Design of the MeCP2 deletion series a, Diagram of the genomic DNA sequences encoding WT and ΔNIC MeCP2, showing the retention of the extreme N-terminal amino acids encoded in exons 1 and 2 and the first 10 bp of exon 3, the deletion of the N- and C-terminal regions, the replacement of the intervening region with a linker and SV40 NLS, and the addition of the C-terminal EGFP tag. Colour key: 5’UTR=white, MBD=blue, NID=pink, other MeCP2 coding regions=grey, SV40 NLS=orange, linkers=dark grey and EGFP=green. b, The N-terminal ends of the sequences of all three truncated proteins (e1 and e2 isoforms) showing the fusion of the extreme N-terminal amino acids to the MBD (starting with P72). c, d, Protein sequence alignment of the MBD ( c ) and NID ( d ) regions using ClustalWS, shaded according to BLOSUM62 score. Both alignments are annotated with RTT-causing missense mutations 31 (red), activity-dependent phosphorylation sites 29 , 32 , 33 (orange), sequence conservation, interaction domains and known 34 /predicted 35 structure. Interaction sites: methyl-DNA binding (residues 78-162 13 ), AT hook 1 (residues 183-195 36 ), AT hook 2 (residues 257-272 28 ), NCoR/SMRT binding (residues 285-309 5 ). The bipartite nuclear localisation signal (NLS) is also shown (residues 253-256 and 266-271). The regions retained in ΔNIC are: MBD resides 72-173 (highlighted by the blue shading in panel c ) and NID resides 272-312 (highlighted by the pink shading panel d). Residue numbers correspond to that of mammalian e2 isoforms.

    Journal: Nature

    Article Title: Radically truncated MeCP2 rescues Rett syndrome-like neurological defects

    doi: 10.1038/nature24058

    Figure Lengend Snippet: Design of the MeCP2 deletion series a, Diagram of the genomic DNA sequences encoding WT and ΔNIC MeCP2, showing the retention of the extreme N-terminal amino acids encoded in exons 1 and 2 and the first 10 bp of exon 3, the deletion of the N- and C-terminal regions, the replacement of the intervening region with a linker and SV40 NLS, and the addition of the C-terminal EGFP tag. Colour key: 5’UTR=white, MBD=blue, NID=pink, other MeCP2 coding regions=grey, SV40 NLS=orange, linkers=dark grey and EGFP=green. b, The N-terminal ends of the sequences of all three truncated proteins (e1 and e2 isoforms) showing the fusion of the extreme N-terminal amino acids to the MBD (starting with P72). c, d, Protein sequence alignment of the MBD ( c ) and NID ( d ) regions using ClustalWS, shaded according to BLOSUM62 score. Both alignments are annotated with RTT-causing missense mutations 31 (red), activity-dependent phosphorylation sites 29 , 32 , 33 (orange), sequence conservation, interaction domains and known 34 /predicted 35 structure. Interaction sites: methyl-DNA binding (residues 78-162 13 ), AT hook 1 (residues 183-195 36 ), AT hook 2 (residues 257-272 28 ), NCoR/SMRT binding (residues 285-309 5 ). The bipartite nuclear localisation signal (NLS) is also shown (residues 253-256 and 266-271). The regions retained in ΔNIC are: MBD resides 72-173 (highlighted by the blue shading in panel c ) and NID resides 272-312 (highlighted by the pink shading panel d). Residue numbers correspond to that of mammalian e2 isoforms.

    Article Snippet: MeCP2 localisation and TBL1X-mCherry recruitment assay NIH-3T3 cells were seeded on coverslips in 6-well plates (25,000 cells per well) and transfected with 2 μg plasmid DNA (pEGFPN1-MeCP2 alone or pEGFPN1-MeCP2 and pmCherry-TBL1X ) using JetPEI (PolyPlus Transfection).

    Techniques: Sequencing, Activity Assay, Binding Assay

    Characterization of the FISC system. a Cre-catalyzed DNA recombination with FISC system using fluorescence imaging and flow cytometry. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169 (P hCMV -CreN59-L9-Coh2-NES-pA), pXY177 (P FRLd -NLS-DocS-L9-CreC60-pA), and pDL78 (P hCMV - loxP -STOP- loxP -EGFP-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) each day for 2 days, and expression of the reporter protein EGFP was visualized by fluorescence microscopy and by flow cytometry at 48 h after the first illumination. Representative images from n = 5 biological replicates. Scale bar, 250 μm. Graph bars represent mean ± SD of n = 3 biological replicates. b Cre-catalyzed DNA recombination with FISC system using luciferase assay. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169, pXY177, and pXY185 (P hCMV - loxP -STOP- loxP -Luciferase-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated as described in ( a ), and bioluminescence measurements were taken at 48 h after the first illumination ( n = 3 independent experiments). c Assessment of illumination-intensity-dependent FISC system activity. 6 × 10 4 HEK-293 cells were cotransfected with pXY137, pXY169, pXY177, and pGY125 (SEAP reporter plasmid) at a 10:1:1:20 (w/w/w/w) ratio and illuminated with FRL for 6 h each day for 2 days at eight different light intensities (0–5 mW cm −2 ); SEAP levels were profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. d Exposure-time-dependent FISC system activity. With the same plasmid ratios as in ( c ), we illuminated transfected cells with FRL (1.5 mW cm −2 , 730 nm) for different time periods (0–120 h). SEAP expression was profiled in the cell culture supernatant at 120 h after initial illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. e FISC-induced SEAP expression in multiple mammalian cell lines. Four different mammalian cell lines were cotransfected and illuminated as described in ( c ), and SEAP expression in the culture supernatant was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Evaluation of the spatial resolution for FISC-mediated transgene expression. A monolayer comprising HEK-293 cells was cotransfected with pXY137, pXY169, pXY177, and pDL78 (EGFP reporter plasmid) at a ratio of a 10:1:1:20 (w/w/w/w), and illuminated as described in ( a ), but through a photomask (schematic, left) with a 6.5 mm slit, and fluorescence microscopy based analysis of the corresponding pattern of EGFP expression at 48 h after the first illumination (right). Representative images from n = 2 biological replicates. b – e Data represent the mean ± SD. Source data for this figure are available in the Source data file.

    Journal: Nature Communications

    Article Title: A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice

    doi: 10.1038/s41467-020-17530-9

    Figure Lengend Snippet: Characterization of the FISC system. a Cre-catalyzed DNA recombination with FISC system using fluorescence imaging and flow cytometry. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169 (P hCMV -CreN59-L9-Coh2-NES-pA), pXY177 (P FRLd -NLS-DocS-L9-CreC60-pA), and pDL78 (P hCMV - loxP -STOP- loxP -EGFP-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) each day for 2 days, and expression of the reporter protein EGFP was visualized by fluorescence microscopy and by flow cytometry at 48 h after the first illumination. Representative images from n = 5 biological replicates. Scale bar, 250 μm. Graph bars represent mean ± SD of n = 3 biological replicates. b Cre-catalyzed DNA recombination with FISC system using luciferase assay. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169, pXY177, and pXY185 (P hCMV - loxP -STOP- loxP -Luciferase-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated as described in ( a ), and bioluminescence measurements were taken at 48 h after the first illumination ( n = 3 independent experiments). c Assessment of illumination-intensity-dependent FISC system activity. 6 × 10 4 HEK-293 cells were cotransfected with pXY137, pXY169, pXY177, and pGY125 (SEAP reporter plasmid) at a 10:1:1:20 (w/w/w/w) ratio and illuminated with FRL for 6 h each day for 2 days at eight different light intensities (0–5 mW cm −2 ); SEAP levels were profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. d Exposure-time-dependent FISC system activity. With the same plasmid ratios as in ( c ), we illuminated transfected cells with FRL (1.5 mW cm −2 , 730 nm) for different time periods (0–120 h). SEAP expression was profiled in the cell culture supernatant at 120 h after initial illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. e FISC-induced SEAP expression in multiple mammalian cell lines. Four different mammalian cell lines were cotransfected and illuminated as described in ( c ), and SEAP expression in the culture supernatant was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Evaluation of the spatial resolution for FISC-mediated transgene expression. A monolayer comprising HEK-293 cells was cotransfected with pXY137, pXY169, pXY177, and pDL78 (EGFP reporter plasmid) at a ratio of a 10:1:1:20 (w/w/w/w), and illuminated as described in ( a ), but through a photomask (schematic, left) with a 6.5 mm slit, and fluorescence microscopy based analysis of the corresponding pattern of EGFP expression at 48 h after the first illumination (right). Representative images from n = 2 biological replicates. b – e Data represent the mean ± SD. Source data for this figure are available in the Source data file.

    Article Snippet: Briefly, 6 × 104 cells were plated per well in a 24-well cell culture plate and cultured for 16 h. The cells were subsequently incubated for 6 h with 50 µL of a 3:1 PEI: DNA mixture (w/w) (polyethyleneimine, MW 40,000, stock solution 1 mg mL−1 in ddH2 O; Polysciences; Cat. no. 24765) containing 0.32 μg of total plasmid DNA for FISC system.

    Techniques: Fluorescence, Imaging, Flow Cytometry, Expressing, Microscopy, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Cell Culture

    AAV delivery of FISC-mediated DNA recombination in transgenic Cre-tdTomato reporter mice. a Schematic depicting the genetic configuration AAV vectors for the FISC DNA recombination system. b Schematic of working principle for transgenic Cre-tdTomato reporter mice in which the loxP -flanked STOP cassette can be excised by Cre recombinase to allow Cre reporter (tdTomato) expression. c Schematic representation of the experimental procedure for FISC-mediated DNA recombination activity in mice. d Representative images of the tdTomato fluorescence in the AAV-transducted reporter mice ( n = 3 mice/group). e The fluorescence measurements of the tdTomato expression shown in ( d ) ( n = 3 mice/group). f Representative images of the tdTomato fluorescence in isolated liver tissue from the AAV-transducted mice shown in ( d ) ( n = 3 mice/group). Scale bar, 1 cm. g Bioluminescence tdTomato expression shown in ( f ) ( n = 3 mice/group). h , i qRT-PCR ( h ) and Western blot ( i ) analysis of tdTomato in isolated liver tissue shown in ( f ) ( n = 3 mice/group). Western blotting images are representative of three mice from two independent experiments. j Representative fluorescence images of the liver sections of the transgenic Cre-tdTomato reporter mice shown in ( f ) ( n = 5 biological replicates. Scale bar, 250 μm). Data in ( e , g , h ) represent the mean ± SEM. Source data for this figure are available in the Source data file.

    Journal: Nature Communications

    Article Title: A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice

    doi: 10.1038/s41467-020-17530-9

    Figure Lengend Snippet: AAV delivery of FISC-mediated DNA recombination in transgenic Cre-tdTomato reporter mice. a Schematic depicting the genetic configuration AAV vectors for the FISC DNA recombination system. b Schematic of working principle for transgenic Cre-tdTomato reporter mice in which the loxP -flanked STOP cassette can be excised by Cre recombinase to allow Cre reporter (tdTomato) expression. c Schematic representation of the experimental procedure for FISC-mediated DNA recombination activity in mice. d Representative images of the tdTomato fluorescence in the AAV-transducted reporter mice ( n = 3 mice/group). e The fluorescence measurements of the tdTomato expression shown in ( d ) ( n = 3 mice/group). f Representative images of the tdTomato fluorescence in isolated liver tissue from the AAV-transducted mice shown in ( d ) ( n = 3 mice/group). Scale bar, 1 cm. g Bioluminescence tdTomato expression shown in ( f ) ( n = 3 mice/group). h , i qRT-PCR ( h ) and Western blot ( i ) analysis of tdTomato in isolated liver tissue shown in ( f ) ( n = 3 mice/group). Western blotting images are representative of three mice from two independent experiments. j Representative fluorescence images of the liver sections of the transgenic Cre-tdTomato reporter mice shown in ( f ) ( n = 5 biological replicates. Scale bar, 250 μm). Data in ( e , g , h ) represent the mean ± SEM. Source data for this figure are available in the Source data file.

    Article Snippet: Briefly, 6 × 104 cells were plated per well in a 24-well cell culture plate and cultured for 16 h. The cells were subsequently incubated for 6 h with 50 µL of a 3:1 PEI: DNA mixture (w/w) (polyethyleneimine, MW 40,000, stock solution 1 mg mL−1 in ddH2 O; Polysciences; Cat. no. 24765) containing 0.32 μg of total plasmid DNA for FISC system.

    Techniques: Transgenic Assay, Mouse Assay, Expressing, Activity Assay, Fluorescence, Isolation, Quantitative RT-PCR, Western Blot

    FISC-induced DNA recombination in BALB/c wild-type mice. a Schematic showing the experimental procedure of light-induced DNA recombination activity in mice. b Comparison of the FISC system with the CRY2-Cre and PA-Cre systems using in vivo bioluminescence imaging. The BALB/c mice were transiently hydrodynamically injected (tail vein) with an iteration of the FISC system comprising three plasmids [pXY137/pXY237 (pA-CreC60-DocS-NLS-P FRLd -Space3-P hCMV -CreN59-Coh2-P2A-ZeoR-pA)/pXY185 (luciferase reporter plasmid) at a ratio of 1.5:1:1 (w/w/w)], or the CRY2-Cre, or the PA-Cre system or only the luciferase reporter plasmid pXY185 as control. At 8 h after the injection, the mice were illuminated with FRL (20 mW cm −2 , 730 nm) or blue light (BL, 20 mW cm −2 , 460 nm) for 12 h (15 min on, 15 min off, alternating) or maintained in the dark, followed by imaging at 12 h after light illumination. c Bioluminescence measurements of the BALB/c mice shown in ( b ) (Control: n = 3, FISC system: n = 4, CRY2-Cre and PA-Cre: n = 5. Data present the mean ± SEM). See Supplementary Table 4 for detailed description of genetic components for each optogenetic system. Source data for this figure are available in the Source data file.

    Journal: Nature Communications

    Article Title: A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice

    doi: 10.1038/s41467-020-17530-9

    Figure Lengend Snippet: FISC-induced DNA recombination in BALB/c wild-type mice. a Schematic showing the experimental procedure of light-induced DNA recombination activity in mice. b Comparison of the FISC system with the CRY2-Cre and PA-Cre systems using in vivo bioluminescence imaging. The BALB/c mice were transiently hydrodynamically injected (tail vein) with an iteration of the FISC system comprising three plasmids [pXY137/pXY237 (pA-CreC60-DocS-NLS-P FRLd -Space3-P hCMV -CreN59-Coh2-P2A-ZeoR-pA)/pXY185 (luciferase reporter plasmid) at a ratio of 1.5:1:1 (w/w/w)], or the CRY2-Cre, or the PA-Cre system or only the luciferase reporter plasmid pXY185 as control. At 8 h after the injection, the mice were illuminated with FRL (20 mW cm −2 , 730 nm) or blue light (BL, 20 mW cm −2 , 460 nm) for 12 h (15 min on, 15 min off, alternating) or maintained in the dark, followed by imaging at 12 h after light illumination. c Bioluminescence measurements of the BALB/c mice shown in ( b ) (Control: n = 3, FISC system: n = 4, CRY2-Cre and PA-Cre: n = 5. Data present the mean ± SEM). See Supplementary Table 4 for detailed description of genetic components for each optogenetic system. Source data for this figure are available in the Source data file.

    Article Snippet: Briefly, 6 × 104 cells were plated per well in a 24-well cell culture plate and cultured for 16 h. The cells were subsequently incubated for 6 h with 50 µL of a 3:1 PEI: DNA mixture (w/w) (polyethyleneimine, MW 40,000, stock solution 1 mg mL−1 in ddH2 O; Polysciences; Cat. no. 24765) containing 0.32 μg of total plasmid DNA for FISC system.

    Techniques: Mouse Assay, Activity Assay, In Vivo, Imaging, Injection, Luciferase, Plasmid Preparation

    Design and optimization of the far-red light-induced split Cre- loxP system (FISC system). a Schematic representation of the FISC system. Cre recombinase was split into two fragments: CreN59 (residues 1–59) fused to Coh2 driven by a constitutive promoter (P hCMV ) and CreC60 (residues 60–343) fused to DocS driven by the far-red light (FRL, 730 nm)-inducible promoter (P FRLx ). Upon FRL illumination, the photoreceptor BphS is activated to convert intracellular guanylate triphosphate (GTP) into cyclic diguanylate monophosphate (c-di-GMP). The cytosolic c-di-GMP production induces binding of the far-red light-dependent transactivator FRTA (p65-VP64-BldD) to its synthetic promoter P FRLx to drive DocS-CreC60 expression. Consequently, the catalytic activities of Cre recombinase can be restored once the two Cre fragments assemble based on affinity interactions of their respective Coh2 and DocS fusion domains, enabling to excise DNA sequences flanked by loxP sites. b Schematic depicting the genetic configuration of constructs used in the FISC system. pA, polyadenylation signals; YhjH, the bacterial c-di-GMP phosphodiesterase; DocS, dockerin S from C. thermocellum complexed with Coh2; L 1-11 , different linkers from 1 to 11 (Supplementary Table 1 ), Coh2, an anchoring protein from C. thermocellum ; loxP , the specific Cre recombinase binding site; STOP, a terminator containing pA to prevent transcription; GOI, gene of interest. c Schematic depicting different genetic configurations of the FRL-inducible promoters P FRLx . 3*whiG, three copies of BldD-specific binding sequence; P hCMVmin , minimal version of P hCMV ; P min , minimal eukaryotic promoter; TATA, minimal eukaryotic promoter with only TATA box. d Schematic depicting the time schedule for the FISC experimental procedure with mammalian cells. e Optimization of the different transfection amounts for CreN59-Coh2 expression and light-inducible P FRLd -driven DocS-CreC60 expression. HEK-293 (6 × 10 4 ) cells were cotransfected with pXY137 (P hCMV -p65-VP64-BldD-pA::P hCMV -BphS-P2A-YhjH-pA, 100 ng), pGY125 (P hCMV - loxP -STOP- loxP -SEAP-pA, 200 ng), pXY110 (P hCMV -CreN59-L0-Coh2-NES-pA) and pXY133 (P FRLd -NLS-DocS-L0-CreC60-pA) from 5 to 100 ng at a ratio of 1:1 (w/w), and then illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) once each day for 2 days. SEAP expression in the culture supernatants was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Optimization of the different linkers (L1–L11) between the CreN59 and Coh2 domains, as well as the CreC60 and DocS domains. The 6 × 10 4 HEK-293 cells per well were cotransfected with pXY137 (100 ng), pGY125 (200 ng), and Docs-CreC60 and Coh2-CreN59 with different combinations of the linkers (10 ng/10 ng) (Supplementary Table 2 ); these were illuminated as described in e , followed by SEAP expression in the culture supernatants profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame in ( e , f ) marks the best-performing condition. e , f Data represent the mean ± SD. Source data for this figure are available in the Source data file.

    Journal: Nature Communications

    Article Title: A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice

    doi: 10.1038/s41467-020-17530-9

    Figure Lengend Snippet: Design and optimization of the far-red light-induced split Cre- loxP system (FISC system). a Schematic representation of the FISC system. Cre recombinase was split into two fragments: CreN59 (residues 1–59) fused to Coh2 driven by a constitutive promoter (P hCMV ) and CreC60 (residues 60–343) fused to DocS driven by the far-red light (FRL, 730 nm)-inducible promoter (P FRLx ). Upon FRL illumination, the photoreceptor BphS is activated to convert intracellular guanylate triphosphate (GTP) into cyclic diguanylate monophosphate (c-di-GMP). The cytosolic c-di-GMP production induces binding of the far-red light-dependent transactivator FRTA (p65-VP64-BldD) to its synthetic promoter P FRLx to drive DocS-CreC60 expression. Consequently, the catalytic activities of Cre recombinase can be restored once the two Cre fragments assemble based on affinity interactions of their respective Coh2 and DocS fusion domains, enabling to excise DNA sequences flanked by loxP sites. b Schematic depicting the genetic configuration of constructs used in the FISC system. pA, polyadenylation signals; YhjH, the bacterial c-di-GMP phosphodiesterase; DocS, dockerin S from C. thermocellum complexed with Coh2; L 1-11 , different linkers from 1 to 11 (Supplementary Table 1 ), Coh2, an anchoring protein from C. thermocellum ; loxP , the specific Cre recombinase binding site; STOP, a terminator containing pA to prevent transcription; GOI, gene of interest. c Schematic depicting different genetic configurations of the FRL-inducible promoters P FRLx . 3*whiG, three copies of BldD-specific binding sequence; P hCMVmin , minimal version of P hCMV ; P min , minimal eukaryotic promoter; TATA, minimal eukaryotic promoter with only TATA box. d Schematic depicting the time schedule for the FISC experimental procedure with mammalian cells. e Optimization of the different transfection amounts for CreN59-Coh2 expression and light-inducible P FRLd -driven DocS-CreC60 expression. HEK-293 (6 × 10 4 ) cells were cotransfected with pXY137 (P hCMV -p65-VP64-BldD-pA::P hCMV -BphS-P2A-YhjH-pA, 100 ng), pGY125 (P hCMV - loxP -STOP- loxP -SEAP-pA, 200 ng), pXY110 (P hCMV -CreN59-L0-Coh2-NES-pA) and pXY133 (P FRLd -NLS-DocS-L0-CreC60-pA) from 5 to 100 ng at a ratio of 1:1 (w/w), and then illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) once each day for 2 days. SEAP expression in the culture supernatants was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Optimization of the different linkers (L1–L11) between the CreN59 and Coh2 domains, as well as the CreC60 and DocS domains. The 6 × 10 4 HEK-293 cells per well were cotransfected with pXY137 (100 ng), pGY125 (200 ng), and Docs-CreC60 and Coh2-CreN59 with different combinations of the linkers (10 ng/10 ng) (Supplementary Table 2 ); these were illuminated as described in e , followed by SEAP expression in the culture supernatants profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame in ( e , f ) marks the best-performing condition. e , f Data represent the mean ± SD. Source data for this figure are available in the Source data file.

    Article Snippet: Briefly, 6 × 104 cells were plated per well in a 24-well cell culture plate and cultured for 16 h. The cells were subsequently incubated for 6 h with 50 µL of a 3:1 PEI: DNA mixture (w/w) (polyethyleneimine, MW 40,000, stock solution 1 mg mL−1 in ddH2 O; Polysciences; Cat. no. 24765) containing 0.32 μg of total plasmid DNA for FISC system.

    Techniques: Binding Assay, Expressing, Construct, Sequencing, Transfection

    Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.

    Journal: ACS synthetic biology

    Article Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9

    doi: 10.1021/acssynbio.7b00050

    Figure Lengend Snippet: Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.

    Article Snippet: ~3.6 μg (~6 nM) of plasmid DNA was added to the preannealed AceCas9:sgRNA, mixed, and aliquoted into prechilled Eppendorf tubes on ice before initiating reactions.

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    Experimental confirmation of the 5′-NNNCC-3′ PAM for AceCas9 on oligo DNA substrates. (A) Cleavage results of double stranded DNA oligos (dsDNA) containing the 5′-NNNCC-3′ PAM. (Left) examination of HEX-labeled targeting DNA strand and its annealed dsDNA product in a nondenaturing gel. (Right) cleavage of dsDNA labeled with fluorophore 6-FAM on its nontargeting strand (red) and cleavage of dsDNA labeled with fluorophore HEX on its targeting strand (green). (B) Cleavage results of dsDNA oligos containing variations in PAM. (Top) Sequences and names of the wild-type and mutant oligo DNA. Mutations are indicated in red. (Bottom) Results of oligo DNA cleavage visualized on a 15% denaturing polyacrylamide gel. The two wild-type (WT) dsDNA are distinguished by the position of the fluorescence labels. The dsDNA with nontargeting strand labeled by 6-FAM is denoted as WT*, whereas that with targeting strand labeled by HEX is denoted as WT. Other dsDNA variants are indicated by nucleotides in red.

    Journal: ACS synthetic biology

    Article Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9

    doi: 10.1021/acssynbio.7b00050

    Figure Lengend Snippet: Experimental confirmation of the 5′-NNNCC-3′ PAM for AceCas9 on oligo DNA substrates. (A) Cleavage results of double stranded DNA oligos (dsDNA) containing the 5′-NNNCC-3′ PAM. (Left) examination of HEX-labeled targeting DNA strand and its annealed dsDNA product in a nondenaturing gel. (Right) cleavage of dsDNA labeled with fluorophore 6-FAM on its nontargeting strand (red) and cleavage of dsDNA labeled with fluorophore HEX on its targeting strand (green). (B) Cleavage results of dsDNA oligos containing variations in PAM. (Top) Sequences and names of the wild-type and mutant oligo DNA. Mutations are indicated in red. (Bottom) Results of oligo DNA cleavage visualized on a 15% denaturing polyacrylamide gel. The two wild-type (WT) dsDNA are distinguished by the position of the fluorescence labels. The dsDNA with nontargeting strand labeled by 6-FAM is denoted as WT*, whereas that with targeting strand labeled by HEX is denoted as WT. Other dsDNA variants are indicated by nucleotides in red.

    Article Snippet: ~3.6 μg (~6 nM) of plasmid DNA was added to the preannealed AceCas9:sgRNA, mixed, and aliquoted into prechilled Eppendorf tubes on ice before initiating reactions.

    Techniques: Labeling, Mutagenesis, Fluorescence

    Identification and purification of the Type II-C Cas9 from A. cellulolyticus (AceCas9). (A) (Top) CRISPR locus in A. cellulolyticus 11B and the sequences of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Black rectangles (R), indicate consensus direct repeat sequence and colored diamonds (S) indicate spacer sequences. The red arrow indicates the putative transcription direction for tracrRNA. (Bottom) Schematic representation of R-loop formation between the dsDNA target and the constructed single guide RNA (sgRNA). The guide region of sgRNA (or spacer) is shown in red and base pairs with the targeting DNA strand of the protospacer DNA. Four guide sequences ranging from 20-nts to 26-nts used in this study are listed. (B) (Top) Domain organization for AceCas9. Conserved catalytic residues are indicated in red. RuvC refers to the RuvC nuclease domain (dark green), ABH refers to the Arginine-rich Bridge Helix (light green), REC refers to the heteroduplex Recognition lobe (gray), HNH refers to the HNH nuclease domain (dark blue), PID refers to the PAM-interacting domains and is composed of 3 segments: β-hairpin (β-H, orange), Topo-homology domain (TOPO, blue), and the C-terminal domain (CTD, light blue). (Bottom left) SDS-PAGE analysis of the wild-type (WT) and H591A mutant AceCas9 following Nickel affinity chromatography (Ni-NTA), ion-exchange (IEC) and size-exclusion chromatography (S200). (Bottom right) Binding of AceCas9:sgRNA or H591A:sgRNA complex to single stranded targeting DNA labeled by HEX. Black line indicates the merged lanes from two different gels.

    Journal: ACS synthetic biology

    Article Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9

    doi: 10.1021/acssynbio.7b00050

    Figure Lengend Snippet: Identification and purification of the Type II-C Cas9 from A. cellulolyticus (AceCas9). (A) (Top) CRISPR locus in A. cellulolyticus 11B and the sequences of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Black rectangles (R), indicate consensus direct repeat sequence and colored diamonds (S) indicate spacer sequences. The red arrow indicates the putative transcription direction for tracrRNA. (Bottom) Schematic representation of R-loop formation between the dsDNA target and the constructed single guide RNA (sgRNA). The guide region of sgRNA (or spacer) is shown in red and base pairs with the targeting DNA strand of the protospacer DNA. Four guide sequences ranging from 20-nts to 26-nts used in this study are listed. (B) (Top) Domain organization for AceCas9. Conserved catalytic residues are indicated in red. RuvC refers to the RuvC nuclease domain (dark green), ABH refers to the Arginine-rich Bridge Helix (light green), REC refers to the heteroduplex Recognition lobe (gray), HNH refers to the HNH nuclease domain (dark blue), PID refers to the PAM-interacting domains and is composed of 3 segments: β-hairpin (β-H, orange), Topo-homology domain (TOPO, blue), and the C-terminal domain (CTD, light blue). (Bottom left) SDS-PAGE analysis of the wild-type (WT) and H591A mutant AceCas9 following Nickel affinity chromatography (Ni-NTA), ion-exchange (IEC) and size-exclusion chromatography (S200). (Bottom right) Binding of AceCas9:sgRNA or H591A:sgRNA complex to single stranded targeting DNA labeled by HEX. Black line indicates the merged lanes from two different gels.

    Article Snippet: ~3.6 μg (~6 nM) of plasmid DNA was added to the preannealed AceCas9:sgRNA, mixed, and aliquoted into prechilled Eppendorf tubes on ice before initiating reactions.

    Techniques: Purification, CRISPR, Sequencing, Construct, SDS Page, Mutagenesis, Affinity Chromatography, Size-exclusion Chromatography, Binding Assay, Labeling

    Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Concentration Assay, Generated, Software, Incubation, Magnetic Beads, DNA Purification, Fluorescence, Purification

    High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: High Throughput Screening Assay, Plasmid Preparation, Next-Generation Sequencing, Isolation, Sonication, Neutralization, Polymerase Chain Reaction, Purification, Concentration Assay

    Design of experiments (DOE) model number 3 for NGS plasmid DNA library preparation: Optimising the reproducibility between different plasmid DNA preparations. ( A ) Two factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time and plasmid DNA sample volume. The evaluated range for each variable is shown. ( B ) There was a total of 11 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were three response variables: the peak fragment size (representing the size at which most DNA fragments are); the peak relative fluorescence unit (RFU); and the concentration of the sample, after magnetic bead purification. 8 plasmid DNA samples were prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. The concentration of the purified samples was also determined using the PicoGreen ® dsDNA quantification assay. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that tagmentation incubation time and DNA sample volume each had a significant effect individually. There was also a significant interaction between the two variables. ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.48, 0.93 and 0.92 for the peak fragment size, peak RFU and concentration respectively. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp, maximum RFU, a concentration of 0.5–5 ng/μl and minimised peak fragment size standard deviation), the optimised conditions suggested by the model are a DNA sample volume of 58.7 nl and a tagmentation incubation time of 12.5 min. ( F ) The Fragment Analyzer outputs for 8 samples, run with a 12.5 min tagmentation incubation time and 50 nl DNA, show that 7/8 of the samples have a peak fragment size of between 200 and 300 bp and an average concentration of 0.68 ng/μl (±0.33 SD). The undetected sample (sample 4) had a concentration below the limit of detection of the Fragment Analyzer (

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 3 for NGS plasmid DNA library preparation: Optimising the reproducibility between different plasmid DNA preparations. ( A ) Two factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time and plasmid DNA sample volume. The evaluated range for each variable is shown. ( B ) There was a total of 11 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were three response variables: the peak fragment size (representing the size at which most DNA fragments are); the peak relative fluorescence unit (RFU); and the concentration of the sample, after magnetic bead purification. 8 plasmid DNA samples were prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. The concentration of the purified samples was also determined using the PicoGreen ® dsDNA quantification assay. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that tagmentation incubation time and DNA sample volume each had a significant effect individually. There was also a significant interaction between the two variables. ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.48, 0.93 and 0.92 for the peak fragment size, peak RFU and concentration respectively. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp, maximum RFU, a concentration of 0.5–5 ng/μl and minimised peak fragment size standard deviation), the optimised conditions suggested by the model are a DNA sample volume of 58.7 nl and a tagmentation incubation time of 12.5 min. ( F ) The Fragment Analyzer outputs for 8 samples, run with a 12.5 min tagmentation incubation time and 50 nl DNA, show that 7/8 of the samples have a peak fragment size of between 200 and 300 bp and an average concentration of 0.68 ng/μl (±0.33 SD). The undetected sample (sample 4) had a concentration below the limit of detection of the Fragment Analyzer (

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Generated, Software, Incubation, Fluorescence, Concentration Assay, Purification, Standard Deviation

    Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Incubation, Purification, Concentration Assay, Sequencing

    Design of experiments (DOE) model number 1 for NGS plasmid DNA library preparation: Optimisation of the lower size limit of fragments. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time, plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. The response variable to be optimised was the lower size limit (the lowest size of DNA fragment detected after magnetic bead purification). A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each set of conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design, using JMP ® software. Both bead concentration and tagmentation incubation time has a significant effect on the lower size limit of the fragmented DNA (Log Worth > 2). ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.96, indicating a very good correlation. ( E ) Visualisation of the optimal lower size limit model using the prediction profiler tool in the JMP ® software, shows that the desired lower size limit of less than 300 bp is achieved with a tagmentation incubation time of > 7.5 min and a magnetic bead concentration of between 1.1–1.8x sample volume.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 1 for NGS plasmid DNA library preparation: Optimisation of the lower size limit of fragments. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time, plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. The response variable to be optimised was the lower size limit (the lowest size of DNA fragment detected after magnetic bead purification). A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each set of conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design, using JMP ® software. Both bead concentration and tagmentation incubation time has a significant effect on the lower size limit of the fragmented DNA (Log Worth > 2). ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.96, indicating a very good correlation. ( E ) Visualisation of the optimal lower size limit model using the prediction profiler tool in the JMP ® software, shows that the desired lower size limit of less than 300 bp is achieved with a tagmentation incubation time of > 7.5 min and a magnetic bead concentration of between 1.1–1.8x sample volume.

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Generated, Software, Incubation, Concentration Assay, Magnetic Beads, DNA Purification, Purification

    Preparation of plasmid DNA libraries for NGS using miniaturised gDNA method. ( A ) Eight plasmid DNA samples were prepared according to Labcyte's miniaturised NGS gDNA library preparation method [ 2 ], using the Nextera XT kit. After purification, the samples were run on the Fragment Analyzer to determine the size of the fragments. ( B ) Plots of the Fragment Analyzer data show the peak fragment size for 7/8 of the samples is greater than 400 bp, which is the maximum limit required for sequencing. ( C ) Summary of the data shows variable relative fluorescence units (RFU) for the 8 samples. The concentration of each purified sample, as measured using the PicoGreen dsDNA assay, is within the range required (0.5–5 ng/μl).

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Preparation of plasmid DNA libraries for NGS using miniaturised gDNA method. ( A ) Eight plasmid DNA samples were prepared according to Labcyte's miniaturised NGS gDNA library preparation method [ 2 ], using the Nextera XT kit. After purification, the samples were run on the Fragment Analyzer to determine the size of the fragments. ( B ) Plots of the Fragment Analyzer data show the peak fragment size for 7/8 of the samples is greater than 400 bp, which is the maximum limit required for sequencing. ( C ) Summary of the data shows variable relative fluorescence units (RFU) for the 8 samples. The concentration of each purified sample, as measured using the PicoGreen dsDNA assay, is within the range required (0.5–5 ng/μl).

    Article Snippet: 3.3 Optimisation of plasmid DNA library preparation for NGS using Design of Experiments For sequencing of plasmid DNA using the Illumina ® MiSeq platform, a library of fragments with a mean size of between 200 and 400 bp, each with a concentration of 0.5–5 ng/μl is required.

    Techniques: Plasmid Preparation, Next-Generation Sequencing, Purification, Sequencing, Fluorescence, Concentration Assay, Picogreen Assay

    Analyses of the optimized peptide sequence (Opt-pep) ( A ) Schematic illustration of biotin–NCX1(301–320) (native sequence), biotin–NCX1(Opt-pep) (optimized peptide sequence), biotin–NCX1(301–320) scrambled (control sequence) and an untagged PLM cyt peptide used in pull-down assay in ( B ). ( B ) Pull-down assays with biotin–NCX1(301–320) and biotin–NCX1(Opt-pep) against the untagged PLM cyt peptide. PLM binding was analysed by immunoblotting using anti-PLM. A biotin–NCX1(301–320) scrambled peptide and beads were used as negative controls. ( C ) Binding of biotin–NCX1(301–320) and biotin–NCX1(Opt-pep) was identified by overlaying the peptides on membranes containing 20-mer overlapping pSer 68 -PLM peptides, followed by immunoblotting using HRP-conjugated anti-biotin. Binding of ( D ) biotin–NCX1(235–254) and ( E ) biotin–NCX1(301–320) to pSer 68 -PLM with or without a pre-incubation of Opt-pep. Binding was analysed by immunoblotting using HRP-conjugated anti-biotin. Phosphorylated Ser 68 is underlined and the C-terminal α-helical region is indicated in ( C )–( E ). Incubation with only HRP-conjugated anti-biotin (omitting incubation with the peptides) was used as negative control (right-hand panels in C – E ). ( F ) Alignment of human, rat, mouse and dog NCX1 sequence. Position of the XIP region and the native NCX1 sequence (amino acids 301–320) used for optimization are underlined. Black boxes indicate identical amino acids (DNA Star).

    Journal: Biochemical Journal

    Article Title: Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1)

    doi: 10.1042/BCJ20160465

    Figure Lengend Snippet: Analyses of the optimized peptide sequence (Opt-pep) ( A ) Schematic illustration of biotin–NCX1(301–320) (native sequence), biotin–NCX1(Opt-pep) (optimized peptide sequence), biotin–NCX1(301–320) scrambled (control sequence) and an untagged PLM cyt peptide used in pull-down assay in ( B ). ( B ) Pull-down assays with biotin–NCX1(301–320) and biotin–NCX1(Opt-pep) against the untagged PLM cyt peptide. PLM binding was analysed by immunoblotting using anti-PLM. A biotin–NCX1(301–320) scrambled peptide and beads were used as negative controls. ( C ) Binding of biotin–NCX1(301–320) and biotin–NCX1(Opt-pep) was identified by overlaying the peptides on membranes containing 20-mer overlapping pSer 68 -PLM peptides, followed by immunoblotting using HRP-conjugated anti-biotin. Binding of ( D ) biotin–NCX1(235–254) and ( E ) biotin–NCX1(301–320) to pSer 68 -PLM with or without a pre-incubation of Opt-pep. Binding was analysed by immunoblotting using HRP-conjugated anti-biotin. Phosphorylated Ser 68 is underlined and the C-terminal α-helical region is indicated in ( C )–( E ). Incubation with only HRP-conjugated anti-biotin (omitting incubation with the peptides) was used as negative control (right-hand panels in C – E ). ( F ) Alignment of human, rat, mouse and dog NCX1 sequence. Position of the XIP region and the native NCX1 sequence (amino acids 301–320) used for optimization are underlined. Black boxes indicate identical amino acids (DNA Star).

    Article Snippet: Plasmid DNA The MGC mouse clone BC079673 (NCX1) was cloned into the first reading frame of pAdTrack-cytomegalovirus (CMV) shuttle vector (plasmid 16405, Addgene).

    Techniques: Sequencing, Pull Down Assay, Binding Assay, Incubation, Negative Control

    Confirmation of a direct PLM–NCX1 interaction ( A ) NCX1 and PLM were analysed in cytoplasmic and membrane fractions isolated from rat neonatal cardiomyocytes and LV using anti-NCX1 and anti-PLM antibodies. GAPDH and calsequestrin were used as controls for cytoplasmic and membrane fractions respectively. ( B ) Epitope mapping was performed by overlaying an array of immobilized overlapping 20-mer PLM peptides with anti-PLM (ab76597, left-hand panel). Amino acids in bold were relevant for anti-PLM binding. Immunoblotting without anti-PLM was used as a negative control (right-hand panel). ( C ) Rat LV, ( D ) brain or ( E and F ) lysate from HEK-293 cells co-transfected with NCX1 and PLM or PLM(S68D) was subjected to immunoprecipitation using anti-NCX1. Immunoprecipitates and lysate was immunoblotted with anti-NCX1 and anti-PLM antibodies. A specific anti-NCX1 blocking peptide and non-relevant rabbit IgG were used as negative controls. ( G ) Schematic presentation of biotinylated peptides covering PLM cyt and pSer 68 -PLM cyt (upper panel). The α-helical region is indicated. Immunoblotting analysis of the two biotinylated peptides using HRP-conjugated anti-biotin is shown in the lower panel. ( H ) Pull-down assay with biotin–PLM cyt and biotin–pSer 68 -PLM cyt against recombinant His–TF–NCX1 cyt (containing the cytoplasmic part of NCX1) using monoclonal anti-biotin-conjugated beads. Pull-down of NCX1 was analysed by immunoblotting using anti-NCX1. ( I ) Alignment of the cytoplasmic part of PLM in human, rat, mouse, pig and dog. Black boxes indicate the identical amino acids (DNA Star). Molecular masses are indicated in kDa. Ab, antibody; IP, immunoprecipitation; pep, peptide; Ab block pep, anti-NCX1 blocking peptide.

    Journal: Biochemical Journal

    Article Title: Development of a high-affinity peptide that prevents phospholemman (PLM) inhibition of the sodium/calcium exchanger 1 (NCX1)

    doi: 10.1042/BCJ20160465

    Figure Lengend Snippet: Confirmation of a direct PLM–NCX1 interaction ( A ) NCX1 and PLM were analysed in cytoplasmic and membrane fractions isolated from rat neonatal cardiomyocytes and LV using anti-NCX1 and anti-PLM antibodies. GAPDH and calsequestrin were used as controls for cytoplasmic and membrane fractions respectively. ( B ) Epitope mapping was performed by overlaying an array of immobilized overlapping 20-mer PLM peptides with anti-PLM (ab76597, left-hand panel). Amino acids in bold were relevant for anti-PLM binding. Immunoblotting without anti-PLM was used as a negative control (right-hand panel). ( C ) Rat LV, ( D ) brain or ( E and F ) lysate from HEK-293 cells co-transfected with NCX1 and PLM or PLM(S68D) was subjected to immunoprecipitation using anti-NCX1. Immunoprecipitates and lysate was immunoblotted with anti-NCX1 and anti-PLM antibodies. A specific anti-NCX1 blocking peptide and non-relevant rabbit IgG were used as negative controls. ( G ) Schematic presentation of biotinylated peptides covering PLM cyt and pSer 68 -PLM cyt (upper panel). The α-helical region is indicated. Immunoblotting analysis of the two biotinylated peptides using HRP-conjugated anti-biotin is shown in the lower panel. ( H ) Pull-down assay with biotin–PLM cyt and biotin–pSer 68 -PLM cyt against recombinant His–TF–NCX1 cyt (containing the cytoplasmic part of NCX1) using monoclonal anti-biotin-conjugated beads. Pull-down of NCX1 was analysed by immunoblotting using anti-NCX1. ( I ) Alignment of the cytoplasmic part of PLM in human, rat, mouse, pig and dog. Black boxes indicate the identical amino acids (DNA Star). Molecular masses are indicated in kDa. Ab, antibody; IP, immunoprecipitation; pep, peptide; Ab block pep, anti-NCX1 blocking peptide.

    Article Snippet: Plasmid DNA The MGC mouse clone BC079673 (NCX1) was cloned into the first reading frame of pAdTrack-cytomegalovirus (CMV) shuttle vector (plasmid 16405, Addgene).

    Techniques: Isolation, Binding Assay, Negative Control, Transfection, Immunoprecipitation, Blocking Assay, Pull Down Assay, Recombinant

    Association of core-GFP mutant derivatives with U17 snoRNP. HeLa cells were transfected with plasmids expressing either core-GFP, or Δ3N, Δ9C, Δ13C, and Δ20C deletion mutants. Proteins extracts were separated by SDS-PAGE and analyzed by Western blotting using anti-GFP MAbs (A) or they were incubated with anti-GFP beads and immunoprecipitated RNAs were analyzed by an RNase A/T1 protection assay using a U17-specific RNA probe (B). Protein and DNA size markers are indicated on the left.

    Journal: Molecular and Cellular Biology

    Article Title: Human H/ACA Small Nucleolar RNPs and Telomerase Share Evolutionarily Conserved Proteins NHP2 and NOP10

    doi:

    Figure Lengend Snippet: Association of core-GFP mutant derivatives with U17 snoRNP. HeLa cells were transfected with plasmids expressing either core-GFP, or Δ3N, Δ9C, Δ13C, and Δ20C deletion mutants. Proteins extracts were separated by SDS-PAGE and analyzed by Western blotting using anti-GFP MAbs (A) or they were incubated with anti-GFP beads and immunoprecipitated RNAs were analyzed by an RNase A/T1 protection assay using a U17-specific RNA probe (B). Protein and DNA size markers are indicated on the left.

    Article Snippet: For transient transfection assays with GFP- or Xpress-tagged constructs, HeLa cells were transfected with plasmid DNA using FuGENE as recommended by the manufacturer (Boehringer Mannheim) and further incubated for 24 to 48 h. Indirect immunofluorescence microscopy was performed essentially as described by Genschik et al. , using purified rabbit anti-hGAR1 antibodies (Abs) ( ) at 1/1,000 dilution, mouse antifibrillarin monoclonal antibody (MAb) 72B9 (a kind gift from J.

    Techniques: Mutagenesis, Transfection, Expressing, SDS Page, Western Blot, Incubation, Immunoprecipitation

    GFP-tagged hNOP10, hNHP2, and dyskerin specifically associate with H/ACA snoRNAs and hTR. HeLa cells were transfected with plasmids expressing hNHP2-GFP, hNOP10-GFP, GFP-dyskerin fusion proteins or GFP alone (vector pβact-ECGFP), and protein extracts were incubated with anti-GFP beads. As an additional control, protein extracts from untransfected HeLa cells were incubated with antifibrillarin beads. Antisense RNA probes specific for U17, U19, U3, and U13 RNAs and hTR were used for RNase A/T1 mapping of RNA from anti-GFP (lanes 3 to 6) and antifibrillarin (lane 7) immunoprecipitates. Lane 1, aliquot of probe; lane 2, mapping of total RNA isolated from nontransfected HeLa cells; for mapping with the hTR probe, five times more total RNA was used. The hTR gel was exposed two times longer than other gels. The sizes of DNA markers in nucleotides are indicated on the left.

    Journal: Molecular and Cellular Biology

    Article Title: Human H/ACA Small Nucleolar RNPs and Telomerase Share Evolutionarily Conserved Proteins NHP2 and NOP10

    doi:

    Figure Lengend Snippet: GFP-tagged hNOP10, hNHP2, and dyskerin specifically associate with H/ACA snoRNAs and hTR. HeLa cells were transfected with plasmids expressing hNHP2-GFP, hNOP10-GFP, GFP-dyskerin fusion proteins or GFP alone (vector pβact-ECGFP), and protein extracts were incubated with anti-GFP beads. As an additional control, protein extracts from untransfected HeLa cells were incubated with antifibrillarin beads. Antisense RNA probes specific for U17, U19, U3, and U13 RNAs and hTR were used for RNase A/T1 mapping of RNA from anti-GFP (lanes 3 to 6) and antifibrillarin (lane 7) immunoprecipitates. Lane 1, aliquot of probe; lane 2, mapping of total RNA isolated from nontransfected HeLa cells; for mapping with the hTR probe, five times more total RNA was used. The hTR gel was exposed two times longer than other gels. The sizes of DNA markers in nucleotides are indicated on the left.

    Article Snippet: For transient transfection assays with GFP- or Xpress-tagged constructs, HeLa cells were transfected with plasmid DNA using FuGENE as recommended by the manufacturer (Boehringer Mannheim) and further incubated for 24 to 48 h. Indirect immunofluorescence microscopy was performed essentially as described by Genschik et al. , using purified rabbit anti-hGAR1 antibodies (Abs) ( ) at 1/1,000 dilution, mouse antifibrillarin monoclonal antibody (MAb) 72B9 (a kind gift from J.

    Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Isolation

    Evaluation of DNA methylation status at a specific sortilin 1 ( SORT1 ) promoter CpG site as an moyamoya disease (MMD) biomarker. (A) DNA methylation status at the specific promoter CpG sites for five candidate genes was determined using pyrosequencing analysis of endothelial colony forming cells (ECFCs) from eight patients with MMD and eight normal controls. Lines inside graphs represent medians. Statistical analyses were performed using the Mann-Whitney U test. DNA methylation status at specific SORT1 CpG sites was determined using ECFC pyrosequencing analysis from an independent sample set of patients with MMD (n=7) and normal controls (n=6). (B) Receiver operating characteristic curves reveal discrimination of patients with MMD from normal controls based on DNA methylation status at a specific SORT1 promoter CpG site. APOD , apolipoprotein D; FAP , fibroblast activation protein alpha; LITAF , lipopolysacchride induced tumor necrosis factor-alpha factor; NUPR1 , nuclear protein 1; AUC, area under the curve. * P

    Journal: Journal of Stroke

    Article Title: Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker

    doi: 10.5853/jos.2018.00962

    Figure Lengend Snippet: Evaluation of DNA methylation status at a specific sortilin 1 ( SORT1 ) promoter CpG site as an moyamoya disease (MMD) biomarker. (A) DNA methylation status at the specific promoter CpG sites for five candidate genes was determined using pyrosequencing analysis of endothelial colony forming cells (ECFCs) from eight patients with MMD and eight normal controls. Lines inside graphs represent medians. Statistical analyses were performed using the Mann-Whitney U test. DNA methylation status at specific SORT1 CpG sites was determined using ECFC pyrosequencing analysis from an independent sample set of patients with MMD (n=7) and normal controls (n=6). (B) Receiver operating characteristic curves reveal discrimination of patients with MMD from normal controls based on DNA methylation status at a specific SORT1 promoter CpG site. APOD , apolipoprotein D; FAP , fibroblast activation protein alpha; LITAF , lipopolysacchride induced tumor necrosis factor-alpha factor; NUPR1 , nuclear protein 1; AUC, area under the curve. * P

    Article Snippet: Briefly, cells grown to 80% to 90% confluence were trypsinized and pelleted, and 5×105 cells were resuspended with 2.0 μg of plasmid DNA (pCMV6-XL5-SORT1 , Origene, Rockville, MD, USA) or pmaxGFP vector and 100 μL of Nucleofector solution in a Nucleofection cuvette, using program U001.

    Techniques: DNA Methylation Assay, Biomarker Assay, MANN-WHITNEY, Activation Assay

    Transcriptional expression of the five candidate genes altered by a DNA methyltransferase inhibitor. Endothelial colony forming cells from normal control N1 were treated with 0, 10, and 20 μM 5-aza-2′-deoxycytidine for 3 days. After treatment with 5-aza-2′-deoxycytidine, mRNA expression was determined by reverse transcription-quantitative polymerase chain reaction. Data are represented as mean±standard deviation (n=3). Statistical analyses were performed using a one-way analysis of variance and Dunnett’s multiple comparison post-tests for comparing treated and untreated cells. (A) Apolipoprotein D ( APOD ), (B) fibroblast activation protein alpha ( FAP ), (C) lipopolysacchride induced tumor necrosis factor-alpha factor ( LITAF ), (D) nuclear protein 1 ( NUPR1 ), and (E) sortilin 1 ( SORT1 ). N, normal control. * P

    Journal: Journal of Stroke

    Article Title: Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker

    doi: 10.5853/jos.2018.00962

    Figure Lengend Snippet: Transcriptional expression of the five candidate genes altered by a DNA methyltransferase inhibitor. Endothelial colony forming cells from normal control N1 were treated with 0, 10, and 20 μM 5-aza-2′-deoxycytidine for 3 days. After treatment with 5-aza-2′-deoxycytidine, mRNA expression was determined by reverse transcription-quantitative polymerase chain reaction. Data are represented as mean±standard deviation (n=3). Statistical analyses were performed using a one-way analysis of variance and Dunnett’s multiple comparison post-tests for comparing treated and untreated cells. (A) Apolipoprotein D ( APOD ), (B) fibroblast activation protein alpha ( FAP ), (C) lipopolysacchride induced tumor necrosis factor-alpha factor ( LITAF ), (D) nuclear protein 1 ( NUPR1 ), and (E) sortilin 1 ( SORT1 ). N, normal control. * P

    Article Snippet: Briefly, cells grown to 80% to 90% confluence were trypsinized and pelleted, and 5×105 cells were resuspended with 2.0 μg of plasmid DNA (pCMV6-XL5-SORT1 , Origene, Rockville, MD, USA) or pmaxGFP vector and 100 μL of Nucleofector solution in a Nucleofection cuvette, using program U001.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Activation Assay

    Promoter CpG sites in the five candidate genes were hypomethylated in endothelial colony forming cells (ECFCs) from patients with moyamoya disease (MMD). The DNA methylation status within the promoter region was analyzed using bisulfite sequencing polymerase chain reaction. Each circle represents CpG dinucleotides. The methylation status of each CpG site is denoted by black (methylated) or white (unmethylated) circles. Stars above the circles indicate the differentially methylated CpG sites in ECFCs, as revealed by DNA methylation microarray analysis. (A) Apolipoprotein D ( APOD ), (B) fibroblast activation protein alpha ( FAP ), (C) lipopolysacchride induced tumor necrosis factor-alpha factor ( LITAF ), (D) nuclear protein 1 ( NUPR1 ), and (E) sortilin 1 ( SORT1 ). N, normal control.

    Journal: Journal of Stroke

    Article Title: Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker

    doi: 10.5853/jos.2018.00962

    Figure Lengend Snippet: Promoter CpG sites in the five candidate genes were hypomethylated in endothelial colony forming cells (ECFCs) from patients with moyamoya disease (MMD). The DNA methylation status within the promoter region was analyzed using bisulfite sequencing polymerase chain reaction. Each circle represents CpG dinucleotides. The methylation status of each CpG site is denoted by black (methylated) or white (unmethylated) circles. Stars above the circles indicate the differentially methylated CpG sites in ECFCs, as revealed by DNA methylation microarray analysis. (A) Apolipoprotein D ( APOD ), (B) fibroblast activation protein alpha ( FAP ), (C) lipopolysacchride induced tumor necrosis factor-alpha factor ( LITAF ), (D) nuclear protein 1 ( NUPR1 ), and (E) sortilin 1 ( SORT1 ). N, normal control.

    Article Snippet: Briefly, cells grown to 80% to 90% confluence were trypsinized and pelleted, and 5×105 cells were resuspended with 2.0 μg of plasmid DNA (pCMV6-XL5-SORT1 , Origene, Rockville, MD, USA) or pmaxGFP vector and 100 μL of Nucleofector solution in a Nucleofection cuvette, using program U001.

    Techniques: DNA Methylation Assay, Methylation Sequencing, Polymerase Chain Reaction, Methylation, Microarray, Activation Assay

    Genotypes of TaCRT - D in partial lines of RIL population using PCR-RFLP. M: DNA ladder 100; O: Opata 85; W: W7984; 1∼26: Partial lines of RIL population.

    Journal: Frontiers in Plant Science

    Article Title: Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.)

    doi: 10.3389/fpls.2017.01557

    Figure Lengend Snippet: Genotypes of TaCRT - D in partial lines of RIL population using PCR-RFLP. M: DNA ladder 100; O: Opata 85; W: W7984; 1∼26: Partial lines of RIL population.

    Article Snippet: Positive clones were selected by colony PCR and the plasmid DNA was extracted with plasmid extraction kit DP-103 (Tiangen, Beijing).

    Techniques: Polymerase Chain Reaction

    Sal I digestion site in the RIL population parents Opata 85 and W7984. To design the PCR-RFLP, TaCTR genes were amplified using the parents (Opata 85 and W7984) as templates, PCR products were digested with restriction enzymes Sal I, and then were analyzed using RFLP. Different bands appeared after both parents were digested ( Figure 12 ). A 898 bp DNA fragment from D genome in Opata 85 by restriction endonuclease digestion and agarose gel, there were 2 Sal I restriction fragments, 593 and 305 bp, respectively.

    Journal: Frontiers in Plant Science

    Article Title: Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.)

    doi: 10.3389/fpls.2017.01557

    Figure Lengend Snippet: Sal I digestion site in the RIL population parents Opata 85 and W7984. To design the PCR-RFLP, TaCTR genes were amplified using the parents (Opata 85 and W7984) as templates, PCR products were digested with restriction enzymes Sal I, and then were analyzed using RFLP. Different bands appeared after both parents were digested ( Figure 12 ). A 898 bp DNA fragment from D genome in Opata 85 by restriction endonuclease digestion and agarose gel, there were 2 Sal I restriction fragments, 593 and 305 bp, respectively.

    Article Snippet: Positive clones were selected by colony PCR and the plasmid DNA was extracted with plasmid extraction kit DP-103 (Tiangen, Beijing).

    Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    The result of restriction enzyme digesting the amplified PCR products between two parents of RIL population. M: DNA ladder 100; O: Opata 85; W: W7984.

    Journal: Frontiers in Plant Science

    Article Title: Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.)

    doi: 10.3389/fpls.2017.01557

    Figure Lengend Snippet: The result of restriction enzyme digesting the amplified PCR products between two parents of RIL population. M: DNA ladder 100; O: Opata 85; W: W7984.

    Article Snippet: Positive clones were selected by colony PCR and the plasmid DNA was extracted with plasmid extraction kit DP-103 (Tiangen, Beijing).

    Techniques: Amplification, Polymerase Chain Reaction

    Schematic diagram depicting a restriction map of a 5.6-kb HindIII fragment of E. faecium JS79 DNA containing two streptogramin A resistance genes and a transposase gene. Bold arrows reflect the orientations of vgaD and vatG and the transposase (IS); black

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Characterization of Two Newly Identified Genes, vgaD and vatG, Conferring Resistance to Streptogramin A in Enterococcus faecium ▿

    doi: 10.1128/AAC.00798-09

    Figure Lengend Snippet: Schematic diagram depicting a restriction map of a 5.6-kb HindIII fragment of E. faecium JS79 DNA containing two streptogramin A resistance genes and a transposase gene. Bold arrows reflect the orientations of vgaD and vatG and the transposase (IS); black

    Article Snippet: A 6-kb HindIII fragment of plasmid DNA from Q-D-resistant E. faecium isolate was sequenced by Macrogen Service Center (Macrogen, Seoul, South Korea) and analyzed using DNAStar software version 5.0 to find open reading frames (ORFs).

    Techniques:

    Southern hybridization to identify vatG -containing E. faecium isolates from healthy humans, swine, poultry stool samples, and chicken meat. (A) Agarose gel electrophoresis of HindIII-digested plasmid DNA from E. faecium isolates. (B) Southern hybridization

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Characterization of Two Newly Identified Genes, vgaD and vatG, Conferring Resistance to Streptogramin A in Enterococcus faecium ▿

    doi: 10.1128/AAC.00798-09

    Figure Lengend Snippet: Southern hybridization to identify vatG -containing E. faecium isolates from healthy humans, swine, poultry stool samples, and chicken meat. (A) Agarose gel electrophoresis of HindIII-digested plasmid DNA from E. faecium isolates. (B) Southern hybridization

    Article Snippet: A 6-kb HindIII fragment of plasmid DNA from Q-D-resistant E. faecium isolate was sequenced by Macrogen Service Center (Macrogen, Seoul, South Korea) and analyzed using DNAStar software version 5.0 to find open reading frames (ORFs).

    Techniques: Hybridization, Agarose Gel Electrophoresis, Plasmid Preparation

    The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Journal: PLoS ONE

    Article Title: The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion

    doi: 10.1371/journal.pone.0205664

    Figure Lengend Snippet: The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Article Snippet: Subsequently, the plasmid DNA was isolated and sequenced using M13 primers ( 5'-TGT AAA ACG ACG GCC AGT-3' (forward), 5'-CAG GAA ACA GCT ATG ACC-3' (reverse)) by MWG eurofins, to ascertain where the transcription start site was on the sequence, as the inserted fragment sequence would begin at the transcription start site, preceded by the universal primer sequence.

    Techniques: Isolation, Sequencing, Activity Assay, In Silico, Polymerase Chain Reaction, Agarose Gel Electrophoresis