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  • 99
    Thermo Fisher hygromycin b selection
    Hygromycin B Selection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore hygromycin b
    Detection of Mcm2 at DS of  oriP  by ChIP. ( A ) Diagram of p818, a 12.6-kb plasmid carrying  oriP  and flanking sequences (light gray), the EBNA1 gene and flanking sequences (black), and hygromycin B-resistance cassette (hph, dark gray). The regions that were amplified by PCR for ChIP are indicated inside the circle. The distal end of the control region, E1 3′, is 560 bp from the edge of FR and 2.1 kb from DS, whereas E1 5′ is 2.1 kb farther away from  oriP . hph is about 6 kb from  oriP.  ( B ) ChIP of chromatin from 293 cells carrying p818. Chromatin (30 μg) was precipitated by using antibodies to Orc2, Mcm2, or EBNA1 or by using nonimmune rabbit IgG, as indicated. Either 1/10th (lanes 2, 4, and 6) or 1/50th (lanes 1, 3, and 5) of the recovered DNA was amplified by PCR to detect the four indicated regions of the plasmid. DNA from the chromatin preparation was tested in lanes 6–8, by using 6.4, 32, and 160 ng, respectively. PCR products were separated by agarose-gel electrophoresis and detected by staining with SYBR Green. A reverse image is shown. M, marker. The specific PCR products ranged from 184 bp (hph) to 435 bp (DS).
    Hygromycin B, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioShop hygromycin b hygb
    Detection of Mcm2 at DS of  oriP  by ChIP. ( A ) Diagram of p818, a 12.6-kb plasmid carrying  oriP  and flanking sequences (light gray), the EBNA1 gene and flanking sequences (black), and hygromycin B-resistance cassette (hph, dark gray). The regions that were amplified by PCR for ChIP are indicated inside the circle. The distal end of the control region, E1 3′, is 560 bp from the edge of FR and 2.1 kb from DS, whereas E1 5′ is 2.1 kb farther away from  oriP . hph is about 6 kb from  oriP.  ( B ) ChIP of chromatin from 293 cells carrying p818. Chromatin (30 μg) was precipitated by using antibodies to Orc2, Mcm2, or EBNA1 or by using nonimmune rabbit IgG, as indicated. Either 1/10th (lanes 2, 4, and 6) or 1/50th (lanes 1, 3, and 5) of the recovered DNA was amplified by PCR to detect the four indicated regions of the plasmid. DNA from the chromatin preparation was tested in lanes 6–8, by using 6.4, 32, and 160 ng, respectively. PCR products were separated by agarose-gel electrophoresis and detected by staining with SYBR Green. A reverse image is shown. M, marker. The specific PCR products ranged from 184 bp (hph) to 435 bp (DS).
    Hygromycin B Hygb, supplied by BioShop, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nacalai hygromycin b hygb
    Improvement of the RdRp-positive cell ratio by polycistronic expression of a selection marker from the most downstream of ORF. (A) Measurement of the RdRp-positive cell ratios. MDCK cells were transfected with unlinearized phCMVK-X-HygR polycistronic expression vectors (X is indicated above a column). Following drug selection in the presence of G418 or <t>HygB</t> (upper and lower panels, respectively) for 2-weeks, indirect immunofluorescence microscopy (top images) and flow cytometry (middle plots and bottom histograms) were carried out using anti-TaV2Apep antibody (FL1, shown in red). In fluorescent images, nuclear DNA and AcGFP were also shown in blue and green, respectively. Axes of flow cytometric plots were indicated in (B) and the parameters were described in the Materials and Methods section. Middle dot plots, cell size (FS Lin) versus TaV2A peptide content (FL1 Log) with a polygon gate and the ratio of TaV2A peptide-positive cells (red). Bottom histograms, the TaV2A-peptide content (FL1 Log) versus cell counts (events), in which TaV2A peptide-positive events (red) are superimposed on the total events (gray). (B) Flow cytometric analyses of untransfected MDCK cells (negative control). (C) MDCK cells were transfected with linearized phCMVK-1a2osHygR and TaV2A peptide-positive clones were isolated by limiting dilution in the presence of HygB ( Supplementary Figure S4 ). After several passages, the clone No. 5 was analyzed by flow cytometer.
    Hygromycin B Hygb, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa hygromycin b
    Generation and validation of human LEDGF/p75 KO cell line. (A) Scheme for PSIP1 gene targeting by homologous recombination. The 2.3 and 2.0 kb arm indicated on the targeting plasmids enable homologous recombination and harbor a puromycin (Puro r ) or <t>hygromycin</t> B resistance (Hyg r ) cassette (c) flanked by loxP sites (arrows). Exon 11 of p52 and p75 are indicated separately as well as the IBD coding region. BamHI restriction sites are indicated (scissors). DT-A denotes the gene encoding for Diphteria toxin A. (B) Schematic overview of KO and intermediates: Nalm-6 wild-type (Nalm +/+ ) contains 2 PSIP1 genes; Nalm +/c clones (cl) 31, 97, 147, contain a puromycin resistance cassette in one allele; Nalm c/c 31 cl 73, contains both a puromycin and a hygromycin B resistance cassette; after CRE-mediated excision cl 1-7 are generated and termed Nalm −/− . (C) Genomic PCR on DNA from different clones. Primer binding sites are indicated in panel A (see also Table S2 ). Indicated bands confirm amplification of a 1.6 kb fragment by primers D and E in full length PSIP1 as shown in Nalm +/c and Nalm +/+ , but not in Nalm c/c and Nalm −/− cl 1. (D) Southern blot on genomic DNA after BamHI digestion. Probe and restriction sites are indicated in panel A. Intact PSIP1 generates a 16.2 kb fragment. After insertion of a resistance cassette a 7.5 kb fragment is generated, after CRE-mediated excision a 14.6 kb fragment is formed indicating KO of a 1.6 kb fragment containing exon 11–14. (E) Western blot analysis for LEDGF protein of whole cell extracts. Marker heights (right), LEDGF/p75 (arrow) and LEDGF KO (arrowhead) are indicated. (F) Nalm-6 cells were transduced with HIV-fLuc. Luciferase expression is shown as percentage relative light units (RLU) per µg protein as compared to Nalm +/c cl 31. (G) In parallel, the number of integrated proviral copies was evaluated for HIV-fLuc. Following transduction, cells were grown for at least 10 days to eliminate non-integrated viral DNA and analyzed by quantitative PCR. (H) HIV-1 integration site distribution analysis. Left panel shows relative number of experimentally derived HIV-1 integration events in genes according to the RefSeq annotation, versus computationally generated matched random control (MRC). The right panel shows integration events occurring ±2 kb around CpG islands as compared with MRC. Average ± standard deviations are shown from experiments performed at least in triplicate.
    Hygromycin B, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris hygromycin b
    Mitochondrial disruption specifically induces the ESRE network. (A) Expression of ESRE-containing genes in worms exposed to 50 μM rotenone or 50 μM antimycin A. Fold changes are normalized to vehicle (DMSO) control. (B) Expression of ESRE-containing and non-ESRE containing genes (red label) after treatment with 1 mM 1,10-phenanthroline (Phe) or with 80 μg/mL <t>hygromycin</t> B (Hygro). irg-1 is a hygromycin-responsive gene used as a control. Fold changes are normalized to vehicle (DMSO) control. Error bars represent SEM.
    Hygromycin B, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen hygromycin b
    Characterization of blasticidin S and <t>hygromycin</t> B resistances in B. burgdorferi . (A) Maps of shuttle vectors pBSV2B and pBSV2H. IR, inverted repeats; cp9 ori , origin or replication of B. burgdorferi plasmid cp9; colE1 ori , E. coli origin of replication; MCS, multicloning site; arr-2 , rifampin resistance gene for selection in E. coli ; P flgB , B. burgdorferi flagellar rod operon promoter; bsd Bb , B. burgdorferi codon-optimized blasticidin S deaminase-encoding gene; hph Bb , B. burgdorferi codon-optimized hygromycin B phosphotransferase-encoding gene. The maps are not drawn to scale. (B) Plate map showing the final antibiotic concentrations used for cross-resistance testing. Each concentration was tested in two adjacent wells. Concentrations routinely used for selection are indicated by the arrow. (C) Schematic representation of color change of the growth medium from red (absence of spirochete growth) to orange/yellow (presence of spirochete growth). A line marks the boundary between growth and no growth in an antibiotic concentration series. The lowest antibiotic concentration that blocked growth was identified as the MIC. (D to H) Susceptibility test of each resistance-carrying strain to various antibiotic concentrations according to the plate layout shown in panel B. The plates were incubated to allow for growth-dependent acidification of the medium and change in phenol red pH indicator color from red to orange and yellow, as depicted in panel C. Images were obtained using colorimetric imaging of the individual plates. MIC boundaries are marked by dark lines, and the MIC values are summarized in Table 3 . The strains used are listed above each image.
    Hygromycin B, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 941 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AG Scientific hygromycin b
    pERα-IRES MDA-MB-231 transfectants: ERα expression via Western immunoblot analysis. A. MDA-MB-231 clones selected for <t>Hygromycin</t> B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-231 parental cell-line represented the negative control. B. Representation of ERα steady state expression values. The values of ERα expression were normalized to tubulin expression, with MCF7 value being one unit.
    Hygromycin B, supplied by AG Scientific, used in various techniques. Bioz Stars score: 93/100, based on 249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amresco hygromycin b
    pERα-IRES MDA-MB-231 transfectants: ERα expression via Western immunoblot analysis. A. MDA-MB-231 clones selected for <t>Hygromycin</t> B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-231 parental cell-line represented the negative control. B. Representation of ERα steady state expression values. The values of ERα expression were normalized to tubulin expression, with MCF7 value being one unit.
    Hygromycin B, supplied by Amresco, used in various techniques. Bioz Stars score: 93/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim hygromycin b
    Southern blot evidence for pMAD10-mediated  URA5 Hc  gene disruption in strain G184AS. Genomic DNAs from transformation recipient strain G184AS (lanes 1) and the four hygromycin B-resistant, uracil-auxotrophic transformants (lanes 2 to 5) were digested with  Cla I, electrophoresed, Southern blotted, and probed with radiolabeled fragments of the  URA5 Hc  gene, the 0.4-kb internal  URA5 Hc  fragment deleted from pMAD10, and the inserted selectable marker  hph . The four uracil auxotrophs (lanes 2 to 5) have lost the native genomic band that hybridizes with the  URA5 Hc  probe and gained a new, larger band due to  hph  insertion that hybridizes with both the  URA5 Hc  and  hph  probes, as well as failing to show any hybridization with the 0.4-kb fragment deleted from the disruption construct pMAD10. Fragment sizes were estimated by comparison to molecular size standards in the ethidium bromide-stained gel.
    Hygromycin B, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 85/100, based on 580 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Carl Roth GmbH hygromycin b
    Expression plasmids for cellulase production. Schematic presentation of the two cellulase expression plasmids used in this study. Both plasmids contain the <t>hygromycin</t> B expression cassette as fungal selection marker followed by either the tef1 or the cDNA1 promoter region and the coding and terminator region of the respective cellulase gene (e.g., the endoglucanase encoding gene cel7b )
    Hygromycin B, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 93/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cellgro hygromycin b
    Expression plasmids for cellulase production. Schematic presentation of the two cellulase expression plasmids used in this study. Both plasmids contain the <t>hygromycin</t> B expression cassette as fungal selection marker followed by either the tef1 or the cDNA1 promoter region and the coding and terminator region of the respective cellulase gene (e.g., the endoglucanase encoding gene cel7b )
    Hygromycin B, supplied by Cellgro, used in various techniques. Bioz Stars score: 91/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences hygromycin b
    A representative colony at 7 days post-hygromycin B selection Phase contrast, 4× magnification. Scale bar represents 1,000 μm.
    Hygromycin B, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Duchefa hygromycin b
    Schematic illustration of the promoter region and the methylation frequencies within the 35S promoter. A , Within the transfer DNA (T-DNA) the resistance marker ( hpt II) is driven by a  nopaline synthase  (NOS) promoter and confers resistance to the antibiotic hygromycin B. The gene of interest (GOI) is driven by a cauliflower mosaic virus promoter (35S). For methylation analysis a 294 bp fragment of the NOS promoter or a 346 bp fragment of the 35S promoter were amplified with the indicated bisulfite primers. Positions of the restriction sites of  Xba I and  Eco RV are indicated.  B , Graphical output of the 35S promoter wide methylation in two consecutive generations of line PNA 1.2 (T 2  and T 3  stage). DNA was isolated from seedlings 15 days post germination (dpg). Filled symbols indicate cytosine methylation, whereas empty symbols indicate a lack of methylation. Red circles represent CG sites, blue squares represent CHG sites and green triangles represent CHH sites, whereas H = A,T or C. Analysis was performed with CyMATE [  127 ]. The upper part illustrates the spatial distribution of the methylation sites within the promoter sequence. Mean cytosine methylation was calculated for the three methylation sites (CG, CHG and CHH) from individually picked clones (±SEM, n = 12 clones).
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    Formedium hygromycin b
    Assaying interactions and relative fitness between kanMX4 and hphEC6 selection markers. a Outline of linearized constructs used in interaction assays. Plasmid elements are not drawn to scale. b Tolerance of yeast strains to combinations of G418 sulfate and hygromycin B. Strains were pre-cultured overnight in 3 mL YM broth with either 200 mg/L G418 disulfate (TLSC001), 200 mg/L <t>hygromycin</t> B (TLSC009), or 200 mg/L of both selection agents (TLSC008 and TLSC010). Pre-cultures were diluted to OD 600 0.1 in fresh YM broth without any selection agent. 2 μL cell suspension of each strain was spotted on solid YM medium with the indicated concentration of G418 disulfate and hygromycin B. Plates were incubated for 2 days at 30 °C and then photographed. c Comparative growth dynamics in MMD broth containing both G418 disulfate and hygromycin B. Error bars indicate one standard deviation. d Fitness equivalence of co-cultivated S. cerevisiae Δput1 strains carrying either the kanMX4 or hphEC6 marker under non-selective conditions. Error bars indicate one standard deviation
    Hygromycin B, supplied by Formedium, used in various techniques. Bioz Stars score: 95/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM hygromycin b
    Concentration of G418 and <t>hygromycin</t> B for the selection of transfected cells. In 24-well plates, 201B7 cells were cultured to confluency and G418 or hygromycin B was added. The cells were examined under the microscope 7 days after the addition of the
    Hygromycin B, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mediatech hygromycin b
    Translational inhibition abolishes the OCTR-1-controlled innate immune responses. ( A ) qRT-PCR of  abu-1 ,  abu-7 ,  abu-8 ,  abu-12 ,  abu-13 ,  abu-14 , and  abu-15  expression in wild-type animals with or without exposure to G418 and in  octr-1 ( ok371 ) animals exposed to G418.  n  = 3; bar graphs correspond to mean ± SEM.  t -tests were performed between N2 + G418 and  octr-1  + G418. *indicates significant difference. ( B ) qRT-PCR of  abu-1 ,  abu-7 ,  abu-8 ,  abu-12 ,  abu-13 ,  abu-14 , and  abu-15  expression in wild-type animals with or without exposure to Hygrocymin B and in  octr-1 ( ok371 ) animals exposed to Hygromycin B.  n  = 3; bar graphs correspond to mean ± SEM.  t -tests were performed between N2 + G418 and  octr-1  + G418. *indicates significant difference. ( C ) Wild-type and  octr-1 ( ok371 ) animals were exposed to NGM/ E. coli  OP50 (control) or NGM/ E. coli  OP50 containing G418 or Hygromycin B, and scored for survival over time. WT + No inhibitor versus  octr-1 ( ok371 )+No inhibitor:  p  = 0.7521; WT + G418 versus  octr-1 ( ok371 )+G418:  p
    Hygromycin B, supplied by Mediatech, used in various techniques. Bioz Stars score: 96/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co hygromycin b
    Translational inhibition abolishes the OCTR-1-controlled innate immune responses. ( A ) qRT-PCR of  abu-1 ,  abu-7 ,  abu-8 ,  abu-12 ,  abu-13 ,  abu-14 , and  abu-15  expression in wild-type animals with or without exposure to G418 and in  octr-1 ( ok371 ) animals exposed to G418.  n  = 3; bar graphs correspond to mean ± SEM.  t -tests were performed between N2 + G418 and  octr-1  + G418. *indicates significant difference. ( B ) qRT-PCR of  abu-1 ,  abu-7 ,  abu-8 ,  abu-12 ,  abu-13 ,  abu-14 , and  abu-15  expression in wild-type animals with or without exposure to Hygrocymin B and in  octr-1 ( ok371 ) animals exposed to Hygromycin B.  n  = 3; bar graphs correspond to mean ± SEM.  t -tests were performed between N2 + G418 and  octr-1  + G418. *indicates significant difference. ( C ) Wild-type and  octr-1 ( ok371 ) animals were exposed to NGM/ E. coli  OP50 (control) or NGM/ E. coli  OP50 containing G418 or Hygromycin B, and scored for survival over time. WT + No inhibitor versus  octr-1 ( ok371 )+No inhibitor:  p  = 0.7521; WT + G418 versus  octr-1 ( ok371 )+G418:  p
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    Merck KGaA hygromycin b
    NAT as a selectable marker that is compatible with paromomycin and/or <t>hygromycin</t> B resistant genes. a Differential interference contrast (DIC) images showing wild type cells, ift54 mutant and rescued cells. Please note that ift54 did not have flagella and flagellar formation was rescued in IFT54- HA expressing cells. Bar, 10 μm. b A diagram showing construct that harbors expression cassettes of IFT54 -HA and NAT . c Expression of IFT54- HA in strains harboring paromomycin resistant gene by using NAT as a selectable marker. The construct listed above was transformed into ift54, which harbors paromomycin resistant gene . The transformants were selected on agar plates supplemented with 10 µg/ml NTC. Cell lysates from randomly picked transformants were subjected to immunoblotting using the indicated antibodies. CDPK3 was used as a loading control. *Denotes non-specific bands. Wild type (WT) and ift54 cells were used as control. d Expression of IFT54- HA in strains harboring paromomycin and hygromycin B resistant genes by using NAT as a selectable marker. wdr92 ::WDR92-YFP strains with both paromomycin and hygromycin B resistant genes were transformed. The expression of IFT54- HA from cells grown on selection plate was examined by immunoblotting. WT and wdr92 ::WDR92-YFP (paro/hygro) cells were used as control
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    Omega Scientific Inc hygromycin b
    NAT as a selectable marker that is compatible with paromomycin and/or <t>hygromycin</t> B resistant genes. a Differential interference contrast (DIC) images showing wild type cells, ift54 mutant and rescued cells. Please note that ift54 did not have flagella and flagellar formation was rescued in IFT54- HA expressing cells. Bar, 10 μm. b A diagram showing construct that harbors expression cassettes of IFT54 -HA and NAT . c Expression of IFT54- HA in strains harboring paromomycin resistant gene by using NAT as a selectable marker. The construct listed above was transformed into ift54, which harbors paromomycin resistant gene . The transformants were selected on agar plates supplemented with 10 µg/ml NTC. Cell lysates from randomly picked transformants were subjected to immunoblotting using the indicated antibodies. CDPK3 was used as a loading control. *Denotes non-specific bands. Wild type (WT) and ift54 cells were used as control. d Expression of IFT54- HA in strains harboring paromomycin and hygromycin B resistant genes by using NAT as a selectable marker. wdr92 ::WDR92-YFP strains with both paromomycin and hygromycin B resistant genes were transformed. The expression of IFT54- HA from cells grown on selection plate was examined by immunoblotting. WT and wdr92 ::WDR92-YFP (paro/hygro) cells were used as control
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    PAA Laboratories hygromycin b
    HA-Rom2(733-1199) induces a different growth phenotype than the full-length protein. (A) In a series of 10-fold dilutions derived from a starting suspension of 5 × 10 6 conidia ml −1 of the indicated strains, aliquots of 3 μl were spotted onto AMM supplemented with the indicated amount of doxycycline (Doxy) and incubated for 48 h at 37°C. AMM agar was additionally supplemented with <t>hygromycin</t> B to avoid loss of the respective construct. (B) A total of 5 × 10 3 conidia ml −1 of the indicated strains were inoculated in the wells on plates in liquid AMM supplemented with no doxycycline (top panels) or 5 μg ml −1 doxycycline (bottom panels). The plates were incubated at 37°C for 16 h. Bar, 250 μm.
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    HA-Rom2(733-1199) induces a different growth phenotype than the full-length protein. (A) In a series of 10-fold dilutions derived from a starting suspension of 5 × 10 6 conidia ml −1 of the indicated strains, aliquots of 3 μl were spotted onto AMM supplemented with the indicated amount of doxycycline (Doxy) and incubated for 48 h at 37°C. AMM agar was additionally supplemented with <t>hygromycin</t> B to avoid loss of the respective construct. (B) A total of 5 × 10 3 conidia ml −1 of the indicated strains were inoculated in the wells on plates in liquid AMM supplemented with no doxycycline (top panels) or 5 μg ml −1 doxycycline (bottom panels). The plates were incubated at 37°C for 16 h. Bar, 250 μm.
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    PhytoTechnology Laboratories hygromycin b
    HA-Rom2(733-1199) induces a different growth phenotype than the full-length protein. (A) In a series of 10-fold dilutions derived from a starting suspension of 5 × 10 6 conidia ml −1 of the indicated strains, aliquots of 3 μl were spotted onto AMM supplemented with the indicated amount of doxycycline (Doxy) and incubated for 48 h at 37°C. AMM agar was additionally supplemented with <t>hygromycin</t> B to avoid loss of the respective construct. (B) A total of 5 × 10 3 conidia ml −1 of the indicated strains were inoculated in the wells on plates in liquid AMM supplemented with no doxycycline (top panels) or 5 μg ml −1 doxycycline (bottom panels). The plates were incubated at 37°C for 16 h. Bar, 250 μm.
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    Roche hygromycin b
    IGR IRES loop 3 mutants bind the 80S ribosomes. ( A ) Assembly of 80S ribosomes on CrPV intergenic region (IGR) IRES loop 3 mutants in rabbit reticulocyte lysate. Radiolabeled CrPV IRES RNAs were incubated in RRL supplemented with <t>hygromycin</t> B for 20 min before separation of initiation complexes on a 15–30% sucrose gradient. Free and 80S-bound IRES complexes are indicated. ( B ) Approximate on- and off-rates of IRES-ribosome binding measured by filter binding. The on-rate experiment measures the association of IRES with ribosomes or ribosomal subunits as a function of time. Pure shrimp ribosomes were used for the on-rate experiment. The off-rate experiment used unlabeled competitor IRES RNA to detect dissociation of IRES from ribosomes as a function of time. Purified yeast subunits were used for the off-rate experiment. DOI: http://dx.doi.org/10.7554/eLife.08146.010
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    Santa Cruz Biotechnology hygromycin b
    IGR IRES loop 3 mutants bind the 80S ribosomes. ( A ) Assembly of 80S ribosomes on CrPV intergenic region (IGR) IRES loop 3 mutants in rabbit reticulocyte lysate. Radiolabeled CrPV IRES RNAs were incubated in RRL supplemented with <t>hygromycin</t> B for 20 min before separation of initiation complexes on a 15–30% sucrose gradient. Free and 80S-bound IRES complexes are indicated. ( B ) Approximate on- and off-rates of IRES-ribosome binding measured by filter binding. The on-rate experiment measures the association of IRES with ribosomes or ribosomal subunits as a function of time. Pure shrimp ribosomes were used for the on-rate experiment. The off-rate experiment used unlabeled competitor IRES RNA to detect dissociation of IRES from ribosomes as a function of time. Purified yeast subunits were used for the off-rate experiment. DOI: http://dx.doi.org/10.7554/eLife.08146.010
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    Stratagene hygromycin b
    IGR IRES loop 3 mutants bind the 80S ribosomes. ( A ) Assembly of 80S ribosomes on CrPV intergenic region (IGR) IRES loop 3 mutants in rabbit reticulocyte lysate. Radiolabeled CrPV IRES RNAs were incubated in RRL supplemented with <t>hygromycin</t> B for 20 min before separation of initiation complexes on a 15–30% sucrose gradient. Free and 80S-bound IRES complexes are indicated. ( B ) Approximate on- and off-rates of IRES-ribosome binding measured by filter binding. The on-rate experiment measures the association of IRES with ribosomes or ribosomal subunits as a function of time. Pure shrimp ribosomes were used for the on-rate experiment. The off-rate experiment used unlabeled competitor IRES RNA to detect dissociation of IRES from ribosomes as a function of time. Purified yeast subunits were used for the off-rate experiment. DOI: http://dx.doi.org/10.7554/eLife.08146.010
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    Thermo Fisher hygromycin b
    New tetracycline- and AID-controlled vectors. (a) Cloning sites of pUHD-AID. Part of the AID tag is shown (the AID tag is 228 amino acid long (~25 kDa)). The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown (except the two BamHI sites). *: termination codon. A puromycin-resistant gene is present in the construct for antibiotic selection in mammalian cells. The complete sequence of pUHD-AID can be found in the Supplemental information. (b) Cloning sites of pRevTRE-AID. The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown. *: termination codon. A hygromycin-resistant gene is expressed from this construct to allow antibiotic selection in mammalian cells. The complete sequence of pRevTRE-AID can be found in the Supplemental information. (c) Workflow for conditional gene inactivation using pUHD-AID or pRevTRE-AID. (1) Silence mutations (changing 3–4 bases) can be introduced into the cDNA to render the cDNA resistant to the CRISPR-Cas9. Alternatively, CRISPR-Cas9 that targets the endogenous gene but not the cDNA (e.g. against a intron-exon boundary) can be used. (2) Cloning sites are shown in panels A and B. (3–4) After cells are transfected with DNA or infected with retroviruses, they are selected with puromycin (pUHD-AID) or <t>hygromycin</t> B (pRevTRE-AID). (5) The cells are then transfected with CRISPR-Cas9 against the gene of interest. Various selection or sorting methods can be used to enrich the transfected cells. (6) Although the entire workflow can be initiated using cells stably expressing TIR1, we found that putting TIR1 into the cells at this stage generated more clones that could effectively respond to IAA. (7) Single clones are isolated and analysed for clones lacking the expression of the endogenous gene of interest, but expressing the AID-tagged version (ideally at a level similar to the endogenous protein) which can be eliminated in the presence of Dox and IAA.
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    Applichem hygromycin b
    New tetracycline- and AID-controlled vectors. (a) Cloning sites of pUHD-AID. Part of the AID tag is shown (the AID tag is 228 amino acid long (~25 kDa)). The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown (except the two BamHI sites). *: termination codon. A puromycin-resistant gene is present in the construct for antibiotic selection in mammalian cells. The complete sequence of pUHD-AID can be found in the Supplemental information. (b) Cloning sites of pRevTRE-AID. The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown. *: termination codon. A hygromycin-resistant gene is expressed from this construct to allow antibiotic selection in mammalian cells. The complete sequence of pRevTRE-AID can be found in the Supplemental information. (c) Workflow for conditional gene inactivation using pUHD-AID or pRevTRE-AID. (1) Silence mutations (changing 3–4 bases) can be introduced into the cDNA to render the cDNA resistant to the CRISPR-Cas9. Alternatively, CRISPR-Cas9 that targets the endogenous gene but not the cDNA (e.g. against a intron-exon boundary) can be used. (2) Cloning sites are shown in panels A and B. (3–4) After cells are transfected with DNA or infected with retroviruses, they are selected with puromycin (pUHD-AID) or <t>hygromycin</t> B (pRevTRE-AID). (5) The cells are then transfected with CRISPR-Cas9 against the gene of interest. Various selection or sorting methods can be used to enrich the transfected cells. (6) Although the entire workflow can be initiated using cells stably expressing TIR1, we found that putting TIR1 into the cells at this stage generated more clones that could effectively respond to IAA. (7) Single clones are isolated and analysed for clones lacking the expression of the endogenous gene of interest, but expressing the AID-tagged version (ideally at a level similar to the endogenous protein) which can be eliminated in the presence of Dox and IAA.
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    PanReac AppliChem hygromycin b
    Synergy is suppressed in mutants resistant to sulphate-transport inhibition. Growth of wild type  S. cerevisiae  BY4741 or isogenic  cys3Δ/met15Δ  or  sul1Δ/sul2Δ  mutants was monitored continuously in YEPD or YNB (for Hygro+DIDS) broths. The appropriate doses of indicated agents used to test synergy were: 50 μg ml −1  paromomycin for wild type and  cys3Δ/met15Δ , 300 μg ml −1  paromomycin for  sul1Δ/sul2Δ , 150 μg ml −1  hygromycin B, 50 μM chromate, 1 mM molybdate, 1 mM DIDS. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.
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    Avantor hygromycin b
    Synergy is suppressed in mutants resistant to sulphate-transport inhibition. Growth of wild type  S. cerevisiae  BY4741 or isogenic  cys3Δ/met15Δ  or  sul1Δ/sul2Δ  mutants was monitored continuously in YEPD or YNB (for Hygro+DIDS) broths. The appropriate doses of indicated agents used to test synergy were: 50 μg ml −1  paromomycin for wild type and  cys3Δ/met15Δ , 300 μg ml −1  paromomycin for  sul1Δ/sul2Δ , 150 μg ml −1  hygromycin B, 50 μM chromate, 1 mM molybdate, 1 mM DIDS. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.
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    Becton Dickinson hygromycin b
    Synergy is suppressed in mutants resistant to sulphate-transport inhibition. Growth of wild type  S. cerevisiae  BY4741 or isogenic  cys3Δ/met15Δ  or  sul1Δ/sul2Δ  mutants was monitored continuously in YEPD or YNB (for Hygro+DIDS) broths. The appropriate doses of indicated agents used to test synergy were: 50 μg ml −1  paromomycin for wild type and  cys3Δ/met15Δ , 300 μg ml −1  paromomycin for  sul1Δ/sul2Δ , 150 μg ml −1  hygromycin B, 50 μM chromate, 1 mM molybdate, 1 mM DIDS. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.
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    Image Search Results


    Detection of Mcm2 at DS of  oriP  by ChIP. ( A ) Diagram of p818, a 12.6-kb plasmid carrying  oriP  and flanking sequences (light gray), the EBNA1 gene and flanking sequences (black), and hygromycin B-resistance cassette (hph, dark gray). The regions that were amplified by PCR for ChIP are indicated inside the circle. The distal end of the control region, E1 3′, is 560 bp from the edge of FR and 2.1 kb from DS, whereas E1 5′ is 2.1 kb farther away from  oriP . hph is about 6 kb from  oriP.  ( B ) ChIP of chromatin from 293 cells carrying p818. Chromatin (30 μg) was precipitated by using antibodies to Orc2, Mcm2, or EBNA1 or by using nonimmune rabbit IgG, as indicated. Either 1/10th (lanes 2, 4, and 6) or 1/50th (lanes 1, 3, and 5) of the recovered DNA was amplified by PCR to detect the four indicated regions of the plasmid. DNA from the chromatin preparation was tested in lanes 6–8, by using 6.4, 32, and 160 ng, respectively. PCR products were separated by agarose-gel electrophoresis and detected by staining with SYBR Green. A reverse image is shown. M, marker. The specific PCR products ranged from 184 bp (hph) to 435 bp (DS).

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

    Article Title: Human DNA replication initiation factors, ORC and MCM, associate with oriP of Epstein-Barr virus

    doi: 10.1073/pnas.181347998

    Figure Lengend Snippet: Detection of Mcm2 at DS of oriP by ChIP. ( A ) Diagram of p818, a 12.6-kb plasmid carrying oriP and flanking sequences (light gray), the EBNA1 gene and flanking sequences (black), and hygromycin B-resistance cassette (hph, dark gray). The regions that were amplified by PCR for ChIP are indicated inside the circle. The distal end of the control region, E1 3′, is 560 bp from the edge of FR and 2.1 kb from DS, whereas E1 5′ is 2.1 kb farther away from oriP . hph is about 6 kb from oriP. ( B ) ChIP of chromatin from 293 cells carrying p818. Chromatin (30 μg) was precipitated by using antibodies to Orc2, Mcm2, or EBNA1 or by using nonimmune rabbit IgG, as indicated. Either 1/10th (lanes 2, 4, and 6) or 1/50th (lanes 1, 3, and 5) of the recovered DNA was amplified by PCR to detect the four indicated regions of the plasmid. DNA from the chromatin preparation was tested in lanes 6–8, by using 6.4, 32, and 160 ng, respectively. PCR products were separated by agarose-gel electrophoresis and detected by staining with SYBR Green. A reverse image is shown. M, marker. The specific PCR products ranged from 184 bp (hph) to 435 bp (DS).

    Article Snippet: A clone of the EBV-negative Burkitt's lymphoma cell line, DG75, that is close to tetraploid and carries p818 at about 50 copies per cell was obtained and maintained in RPMI medium supplemented with 9% FBS, penicillin and streptomycin, and hygromycin B at 300 μg/ml. p818 (Fig. A ) is a 12.6-kb plasmid similar to p201 ( ) but contains 4 kb of additional EBV DNA extending rightward from oriP to the Bam HI site at 13215.

    Techniques: Chromatin Immunoprecipitation, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, Marker

    Working model of single-molecule CRISPR-Cas9 transformation in S. sclerotiorum . The top half of the diagram illustrates the transformation and vector sequence integration events as described in the text. The bottom half of the diagram illustrates the deletion of tandem, direct repeat vector sequences by intrachromosomal recombination during meiosis following self-mating. NHEJ, nonhomologous end joining; DSB, double-stranded break; NLS, nuclear localization sequence; PAM, protospacer adjacent motif; Hph, hygromycin B phosphotransferase; gRNA, guide RNA; Cas9, CRISPR-associated protein 9; RNP, ribonucleoprotein.

    Journal: mBio

    Article Title: Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen Sclerotinia sclerotiorum

    doi: 10.1128/mBio.00567-18

    Figure Lengend Snippet: Working model of single-molecule CRISPR-Cas9 transformation in S. sclerotiorum . The top half of the diagram illustrates the transformation and vector sequence integration events as described in the text. The bottom half of the diagram illustrates the deletion of tandem, direct repeat vector sequences by intrachromosomal recombination during meiosis following self-mating. NHEJ, nonhomologous end joining; DSB, double-stranded break; NLS, nuclear localization sequence; PAM, protospacer adjacent motif; Hph, hygromycin B phosphotransferase; gRNA, guide RNA; Cas9, CRISPR-associated protein 9; RNP, ribonucleoprotein.

    Article Snippet: Transformation mutants were cultured on PDA or regeneration medium (RM; 239.6 g sucrose, 0.5 g yeast extract, 15 g agar/liter or 8 g/liter agar for top agar) supplemented with 100 µg/ml hygromycin B (HygB) (EMD Biosciences, USA) and 50 µg/ml bromophenol blue (Sigma-Aldrich, USA) where indicated.

    Techniques: CRISPR, Transformation Assay, Plasmid Preparation, Sequencing, Non-Homologous End Joining

    Characterization of CRISPR-Cas9-inserted sequences following meiosis. (A) Single-ascospore progeny (+①, +②, -①, and -②) of primary transformant UF1- oah1 -3-6 (wild-type “UF1” background, Ssoah1 target site 3, strain number 6) were assayed for OA production and hygromycin B (HygB) sensitivity on potato dextrose agar (PDA) medium supplemented with bromophenol blue (BPB) and HygB, respectively. Oxalate production (acidification) is indicated by a change in PDA-BPB medium from blue to yellow. Hygromycin B resistance (UF1- oah1 -3-6), tolerance (UF1- oah1 -3-6+①, +②), and sensitivity (UF1, UF1- oah1 -3-6-①, -②) were assayed at 3 and 8 days after inoculation (DAI) of PDA-HygB medium. (B) Long-amplification PCR characterization of target site-inserted sequences in UF1, UF1- oah1 -3-6, and single-ascospore progeny (+①, +②, -①, and -②). Primer pair F and R [ oah1 (C)] for amplification across the Ssoah1 target site, Hyg-P-F and Hyg-T-R [ hph (P-C-T)] for amplification of the entire hph cassette, primer pair Hyg-P-F and Hyg-C-R [ hph (P-C)] for amplification of the TrpC promoter and hph coding sequences, and Hyg-C-F and Hyg-C-R [ hph (C)] for amplification of the hph coding sequence. Primers PycF and PycR specific to Sspyc1 were used as a positive PCR control ( pyc1 ). Primer sequences are given in Table S1 . (C) Global sequence alignment between the transformation vector, pCRISPR-Cas9-TrpC-Hyg, and the insertion sequence obtained from the genome sequence of primary transformant UF1- oah1 -3-6 and its single-ascospore-derived HygB-sensitive progeny UF1- oah1 -3-6-②. Note that three gaps exist among the assembled genomic sequence contigs of UF1- oah1 -3-6 that presumably represent the locations of repeated unassembled sequences. Primer sequences are given in Table S1 .

    Journal: mBio

    Article Title: Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen Sclerotinia sclerotiorum

    doi: 10.1128/mBio.00567-18

    Figure Lengend Snippet: Characterization of CRISPR-Cas9-inserted sequences following meiosis. (A) Single-ascospore progeny (+①, +②, -①, and -②) of primary transformant UF1- oah1 -3-6 (wild-type “UF1” background, Ssoah1 target site 3, strain number 6) were assayed for OA production and hygromycin B (HygB) sensitivity on potato dextrose agar (PDA) medium supplemented with bromophenol blue (BPB) and HygB, respectively. Oxalate production (acidification) is indicated by a change in PDA-BPB medium from blue to yellow. Hygromycin B resistance (UF1- oah1 -3-6), tolerance (UF1- oah1 -3-6+①, +②), and sensitivity (UF1, UF1- oah1 -3-6-①, -②) were assayed at 3 and 8 days after inoculation (DAI) of PDA-HygB medium. (B) Long-amplification PCR characterization of target site-inserted sequences in UF1, UF1- oah1 -3-6, and single-ascospore progeny (+①, +②, -①, and -②). Primer pair F and R [ oah1 (C)] for amplification across the Ssoah1 target site, Hyg-P-F and Hyg-T-R [ hph (P-C-T)] for amplification of the entire hph cassette, primer pair Hyg-P-F and Hyg-C-R [ hph (P-C)] for amplification of the TrpC promoter and hph coding sequences, and Hyg-C-F and Hyg-C-R [ hph (C)] for amplification of the hph coding sequence. Primers PycF and PycR specific to Sspyc1 were used as a positive PCR control ( pyc1 ). Primer sequences are given in Table S1 . (C) Global sequence alignment between the transformation vector, pCRISPR-Cas9-TrpC-Hyg, and the insertion sequence obtained from the genome sequence of primary transformant UF1- oah1 -3-6 and its single-ascospore-derived HygB-sensitive progeny UF1- oah1 -3-6-②. Note that three gaps exist among the assembled genomic sequence contigs of UF1- oah1 -3-6 that presumably represent the locations of repeated unassembled sequences. Primer sequences are given in Table S1 .

    Article Snippet: Transformation mutants were cultured on PDA or regeneration medium (RM; 239.6 g sucrose, 0.5 g yeast extract, 15 g agar/liter or 8 g/liter agar for top agar) supplemented with 100 µg/ml hygromycin B (HygB) (EMD Biosciences, USA) and 50 µg/ml bromophenol blue (Sigma-Aldrich, USA) where indicated.

    Techniques: CRISPR, Amplification, Polymerase Chain Reaction, Sequencing, Transformation Assay, Plasmid Preparation, Derivative Assay

    Extinction of IL2R transgene activity and accompanying DNA methylation of a low-copy-number cell line. ( A ) FACS analysis of IL2R activity at day 0 (in the presence of hygromycin) and at days 8, 16, 23, 28 and 100 after removal of hygromycin. ( B ) Southern blots at different days in culture to determine the extent of DNA methylation at Hpa II restriction sites in the transgene. The top diagram shows the positions of recognition sites for the methylation-sensitive enzyme Hpa II within the IL2R transgene. Horizontal black lines with asterisks at the ends indicate the three products expected from complete digestion with the Hpa II enzyme. The sites protected from digestion due to CpG methylation are indicated as M. The bottom panel shows DNA isolated at different days, digested with the Xba I/ Hpa II restriction enzymes and analyzed by Southern blotting after separation on an agarose gel. The membranes were hybridized with a Bam HI– Xba I probe from the IL2R cDNA, indicated in the top diagram. ( C ) Bisulfite genomic sequencing analysis of a low-copy-number cell line at days 1, 11, 21, 29, 100 and 160. In total, 20 individual clones were sequenced to give the methylation pattern of the 21 CpGs. The percentage of methylated clones for each individual CpG is plotted.

    Journal: The EMBO Journal

    Article Title: Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9

    doi: 10.1038/sj.emboj.7600013

    Figure Lengend Snippet: Extinction of IL2R transgene activity and accompanying DNA methylation of a low-copy-number cell line. ( A ) FACS analysis of IL2R activity at day 0 (in the presence of hygromycin) and at days 8, 16, 23, 28 and 100 after removal of hygromycin. ( B ) Southern blots at different days in culture to determine the extent of DNA methylation at Hpa II restriction sites in the transgene. The top diagram shows the positions of recognition sites for the methylation-sensitive enzyme Hpa II within the IL2R transgene. Horizontal black lines with asterisks at the ends indicate the three products expected from complete digestion with the Hpa II enzyme. The sites protected from digestion due to CpG methylation are indicated as M. The bottom panel shows DNA isolated at different days, digested with the Xba I/ Hpa II restriction enzymes and analyzed by Southern blotting after separation on an agarose gel. The membranes were hybridized with a Bam HI– Xba I probe from the IL2R cDNA, indicated in the top diagram. ( C ) Bisulfite genomic sequencing analysis of a low-copy-number cell line at days 1, 11, 21, 29, 100 and 160. In total, 20 individual clones were sequenced to give the methylation pattern of the 21 CpGs. The percentage of methylated clones for each individual CpG is plotted.

    Article Snippet: Individual hygromycin-resistant colonies were picked after 2–3 weeks of culture in α-MEM plus 2% methocel and 2000 U/ml hygromycin (Calbiochem), and expanded in hygromycin-containing medium (1250 U/ml) for a further 6 days.

    Techniques: Activity Assay, DNA Methylation Assay, Low Copy Number, FACS, Methylation, CpG Methylation Assay, Isolation, Southern Blot, Agarose Gel Electrophoresis, Genomic Sequencing, Clone Assay

    Extinction of IL2R transgene activity over an extended time in culture. ( A ) 6C2 cells carrying 13 copies of the IL2R reporter were grown for extended times without hygromycin selection. For IL2R cell surface activity, the cells were analyzed by flow cytometry at different days. The horizontal bars denote M1 and M2, which define the ranges of IL2R negative and positive cells, respectively. 6C2 cells untransfected with the IL2R construct were used to determine the IL2R negative cells zone on the histograms. ( B ) Quantitative RT–PCR of a 13-copy cell line at days 0, 3, 6, 9, 13, 18 and 20. Each point on the graph represents the amount of IL2R mRNA relative to the folate receptor mRNA per copy of the transgene. IL2R mRNA at day 0 is set to 100.

    Journal: The EMBO Journal

    Article Title: Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9

    doi: 10.1038/sj.emboj.7600013

    Figure Lengend Snippet: Extinction of IL2R transgene activity over an extended time in culture. ( A ) 6C2 cells carrying 13 copies of the IL2R reporter were grown for extended times without hygromycin selection. For IL2R cell surface activity, the cells were analyzed by flow cytometry at different days. The horizontal bars denote M1 and M2, which define the ranges of IL2R negative and positive cells, respectively. 6C2 cells untransfected with the IL2R construct were used to determine the IL2R negative cells zone on the histograms. ( B ) Quantitative RT–PCR of a 13-copy cell line at days 0, 3, 6, 9, 13, 18 and 20. Each point on the graph represents the amount of IL2R mRNA relative to the folate receptor mRNA per copy of the transgene. IL2R mRNA at day 0 is set to 100.

    Article Snippet: Individual hygromycin-resistant colonies were picked after 2–3 weeks of culture in α-MEM plus 2% methocel and 2000 U/ml hygromycin (Calbiochem), and expanded in hygromycin-containing medium (1250 U/ml) for a further 6 days.

    Techniques: Activity Assay, Selection, Flow Cytometry, Cytometry, Construct, Quantitative RT-PCR

    Improvement of the RdRp-positive cell ratio by polycistronic expression of a selection marker from the most downstream of ORF. (A) Measurement of the RdRp-positive cell ratios. MDCK cells were transfected with unlinearized phCMVK-X-HygR polycistronic expression vectors (X is indicated above a column). Following drug selection in the presence of G418 or HygB (upper and lower panels, respectively) for 2-weeks, indirect immunofluorescence microscopy (top images) and flow cytometry (middle plots and bottom histograms) were carried out using anti-TaV2Apep antibody (FL1, shown in red). In fluorescent images, nuclear DNA and AcGFP were also shown in blue and green, respectively. Axes of flow cytometric plots were indicated in (B) and the parameters were described in the Materials and Methods section. Middle dot plots, cell size (FS Lin) versus TaV2A peptide content (FL1 Log) with a polygon gate and the ratio of TaV2A peptide-positive cells (red). Bottom histograms, the TaV2A-peptide content (FL1 Log) versus cell counts (events), in which TaV2A peptide-positive events (red) are superimposed on the total events (gray). (B) Flow cytometric analyses of untransfected MDCK cells (negative control). (C) MDCK cells were transfected with linearized phCMVK-1a2osHygR and TaV2A peptide-positive clones were isolated by limiting dilution in the presence of HygB ( Supplementary Figure S4 ). After several passages, the clone No. 5 was analyzed by flow cytometer.

    Journal: Frontiers in Microbiology

    Article Title: Polycistronic Expression of the Influenza A Virus RNA-Dependent RNA Polymerase by Using the Thosea asigna Virus 2A-Like Self-Processing Sequence

    doi: 10.3389/fmicb.2016.00288

    Figure Lengend Snippet: Improvement of the RdRp-positive cell ratio by polycistronic expression of a selection marker from the most downstream of ORF. (A) Measurement of the RdRp-positive cell ratios. MDCK cells were transfected with unlinearized phCMVK-X-HygR polycistronic expression vectors (X is indicated above a column). Following drug selection in the presence of G418 or HygB (upper and lower panels, respectively) for 2-weeks, indirect immunofluorescence microscopy (top images) and flow cytometry (middle plots and bottom histograms) were carried out using anti-TaV2Apep antibody (FL1, shown in red). In fluorescent images, nuclear DNA and AcGFP were also shown in blue and green, respectively. Axes of flow cytometric plots were indicated in (B) and the parameters were described in the Materials and Methods section. Middle dot plots, cell size (FS Lin) versus TaV2A peptide content (FL1 Log) with a polygon gate and the ratio of TaV2A peptide-positive cells (red). Bottom histograms, the TaV2A-peptide content (FL1 Log) versus cell counts (events), in which TaV2A peptide-positive events (red) are superimposed on the total events (gray). (B) Flow cytometric analyses of untransfected MDCK cells (negative control). (C) MDCK cells were transfected with linearized phCMVK-1a2osHygR and TaV2A peptide-positive clones were isolated by limiting dilution in the presence of HygB ( Supplementary Figure S4 ). After several passages, the clone No. 5 was analyzed by flow cytometer.

    Article Snippet: For stable cell lines, the medium was replaced with selective medium including the neomycin analog (G418) or hygromycin B (HygB) (Nacalai Tesque, Japan).

    Techniques: Expressing, Selection, Marker, Transfection, Immunofluorescence, Microscopy, Flow Cytometry, Cytometry, Negative Control, Clone Assay, Isolation

    Schematic representations of polycistronic RdRp expression vectors. (A) Concatenation of CDSs of RdRp subunits with TaV2A self-processing sequence. The bicistronic PB1-PA ORF is shown as an example. The stop codon ( ∗ ) of the upstream CDS was removed and GS linker-TaV2A peptide CDS was added to the end. Since TaV2A peptide was separated at the Gly-Pro present at the end of the peptide (an arrowhead with a line), the downstream subunit has an additional proline residue at the N-terminal end. Consequently, the original first ATG/methionine codon of the downstream CDS was replaced by proline (M1P substitution, indicated as “Pro” in the PA CDS). (B) CDS order of bi-, tri-, and tetra-cistronic ORFs. PB2, PB1, PA, and the hygromycin B phosphotransferase CDSs (abbreviated as 2, 1, a, and HygR, respectively) were concatenated in-frame with TaV2A peptide (2A) and/or One-STrEP/Twin-strep affinity tag (OS) CDSs ( Schmidt et al., 2013 ). The start and end of each ORF are indicated by open (first ATG) and closed (stop) triangles, respectively. TaV2A peptide-derived proline codons/residues are indicated with “Pro” at the beginning of each downstream CDS. The names (left side) and lengths of ORFs (right side) are indicated. (C) Plasmid map of phCMV1-f1Kpn, a derivative of phCMV1. Additional Kpn I site (indicated as ∗ Kpn I) was created in the f1 ori region. The ORFs exemplified in (B) were inserted between unique Xho I and Not I sites of phCMV1 or phCMV1-f1Kpn and were transcribed to mRNA under the control of CMV promoter (pCMV). The original phCMV1 vector was routinely used for protein expression. The phCMV1-f1Kpn vector was for establishing stable cell lines expressing RdRp.

    Journal: Frontiers in Microbiology

    Article Title: Polycistronic Expression of the Influenza A Virus RNA-Dependent RNA Polymerase by Using the Thosea asigna Virus 2A-Like Self-Processing Sequence

    doi: 10.3389/fmicb.2016.00288

    Figure Lengend Snippet: Schematic representations of polycistronic RdRp expression vectors. (A) Concatenation of CDSs of RdRp subunits with TaV2A self-processing sequence. The bicistronic PB1-PA ORF is shown as an example. The stop codon ( ∗ ) of the upstream CDS was removed and GS linker-TaV2A peptide CDS was added to the end. Since TaV2A peptide was separated at the Gly-Pro present at the end of the peptide (an arrowhead with a line), the downstream subunit has an additional proline residue at the N-terminal end. Consequently, the original first ATG/methionine codon of the downstream CDS was replaced by proline (M1P substitution, indicated as “Pro” in the PA CDS). (B) CDS order of bi-, tri-, and tetra-cistronic ORFs. PB2, PB1, PA, and the hygromycin B phosphotransferase CDSs (abbreviated as 2, 1, a, and HygR, respectively) were concatenated in-frame with TaV2A peptide (2A) and/or One-STrEP/Twin-strep affinity tag (OS) CDSs ( Schmidt et al., 2013 ). The start and end of each ORF are indicated by open (first ATG) and closed (stop) triangles, respectively. TaV2A peptide-derived proline codons/residues are indicated with “Pro” at the beginning of each downstream CDS. The names (left side) and lengths of ORFs (right side) are indicated. (C) Plasmid map of phCMV1-f1Kpn, a derivative of phCMV1. Additional Kpn I site (indicated as ∗ Kpn I) was created in the f1 ori region. The ORFs exemplified in (B) were inserted between unique Xho I and Not I sites of phCMV1 or phCMV1-f1Kpn and were transcribed to mRNA under the control of CMV promoter (pCMV). The original phCMV1 vector was routinely used for protein expression. The phCMV1-f1Kpn vector was for establishing stable cell lines expressing RdRp.

    Article Snippet: For stable cell lines, the medium was replaced with selective medium including the neomycin analog (G418) or hygromycin B (HygB) (Nacalai Tesque, Japan).

    Techniques: Expressing, Sequencing, Derivative Assay, Plasmid Preparation, Stable Transfection

    Generation and validation of human LEDGF/p75 KO cell line. (A) Scheme for PSIP1 gene targeting by homologous recombination. The 2.3 and 2.0 kb arm indicated on the targeting plasmids enable homologous recombination and harbor a puromycin (Puro r ) or hygromycin B resistance (Hyg r ) cassette (c) flanked by loxP sites (arrows). Exon 11 of p52 and p75 are indicated separately as well as the IBD coding region. BamHI restriction sites are indicated (scissors). DT-A denotes the gene encoding for Diphteria toxin A. (B) Schematic overview of KO and intermediates: Nalm-6 wild-type (Nalm +/+ ) contains 2 PSIP1 genes; Nalm +/c clones (cl) 31, 97, 147, contain a puromycin resistance cassette in one allele; Nalm c/c 31 cl 73, contains both a puromycin and a hygromycin B resistance cassette; after CRE-mediated excision cl 1-7 are generated and termed Nalm −/− . (C) Genomic PCR on DNA from different clones. Primer binding sites are indicated in panel A (see also Table S2 ). Indicated bands confirm amplification of a 1.6 kb fragment by primers D and E in full length PSIP1 as shown in Nalm +/c and Nalm +/+ , but not in Nalm c/c and Nalm −/− cl 1. (D) Southern blot on genomic DNA after BamHI digestion. Probe and restriction sites are indicated in panel A. Intact PSIP1 generates a 16.2 kb fragment. After insertion of a resistance cassette a 7.5 kb fragment is generated, after CRE-mediated excision a 14.6 kb fragment is formed indicating KO of a 1.6 kb fragment containing exon 11–14. (E) Western blot analysis for LEDGF protein of whole cell extracts. Marker heights (right), LEDGF/p75 (arrow) and LEDGF KO (arrowhead) are indicated. (F) Nalm-6 cells were transduced with HIV-fLuc. Luciferase expression is shown as percentage relative light units (RLU) per µg protein as compared to Nalm +/c cl 31. (G) In parallel, the number of integrated proviral copies was evaluated for HIV-fLuc. Following transduction, cells were grown for at least 10 days to eliminate non-integrated viral DNA and analyzed by quantitative PCR. (H) HIV-1 integration site distribution analysis. Left panel shows relative number of experimentally derived HIV-1 integration events in genes according to the RefSeq annotation, versus computationally generated matched random control (MRC). The right panel shows integration events occurring ±2 kb around CpG islands as compared with MRC. Average ± standard deviations are shown from experiments performed at least in triplicate.

    Journal: PLoS Pathogens

    Article Title: LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs

    doi: 10.1371/journal.ppat.1002558

    Figure Lengend Snippet: Generation and validation of human LEDGF/p75 KO cell line. (A) Scheme for PSIP1 gene targeting by homologous recombination. The 2.3 and 2.0 kb arm indicated on the targeting plasmids enable homologous recombination and harbor a puromycin (Puro r ) or hygromycin B resistance (Hyg r ) cassette (c) flanked by loxP sites (arrows). Exon 11 of p52 and p75 are indicated separately as well as the IBD coding region. BamHI restriction sites are indicated (scissors). DT-A denotes the gene encoding for Diphteria toxin A. (B) Schematic overview of KO and intermediates: Nalm-6 wild-type (Nalm +/+ ) contains 2 PSIP1 genes; Nalm +/c clones (cl) 31, 97, 147, contain a puromycin resistance cassette in one allele; Nalm c/c 31 cl 73, contains both a puromycin and a hygromycin B resistance cassette; after CRE-mediated excision cl 1-7 are generated and termed Nalm −/− . (C) Genomic PCR on DNA from different clones. Primer binding sites are indicated in panel A (see also Table S2 ). Indicated bands confirm amplification of a 1.6 kb fragment by primers D and E in full length PSIP1 as shown in Nalm +/c and Nalm +/+ , but not in Nalm c/c and Nalm −/− cl 1. (D) Southern blot on genomic DNA after BamHI digestion. Probe and restriction sites are indicated in panel A. Intact PSIP1 generates a 16.2 kb fragment. After insertion of a resistance cassette a 7.5 kb fragment is generated, after CRE-mediated excision a 14.6 kb fragment is formed indicating KO of a 1.6 kb fragment containing exon 11–14. (E) Western blot analysis for LEDGF protein of whole cell extracts. Marker heights (right), LEDGF/p75 (arrow) and LEDGF KO (arrowhead) are indicated. (F) Nalm-6 cells were transduced with HIV-fLuc. Luciferase expression is shown as percentage relative light units (RLU) per µg protein as compared to Nalm +/c cl 31. (G) In parallel, the number of integrated proviral copies was evaluated for HIV-fLuc. Following transduction, cells were grown for at least 10 days to eliminate non-integrated viral DNA and analyzed by quantitative PCR. (H) HIV-1 integration site distribution analysis. Left panel shows relative number of experimentally derived HIV-1 integration events in genes according to the RefSeq annotation, versus computationally generated matched random control (MRC). The right panel shows integration events occurring ±2 kb around CpG islands as compared with MRC. Average ± standard deviations are shown from experiments performed at least in triplicate.

    Article Snippet: At 24 hrs after transfection, cells were seeded into 96-well plates at 103 cells per well, in culture medium supplemented with either 0.2 µg/ml puromycin (BD BioSciences, San Jose, CA, USA) or 0.35 mg/ml of hygromycin B (Clontech, Mountain View, CA, USA).

    Techniques: Homologous Recombination, Generated, Polymerase Chain Reaction, Clone Assay, Binding Assay, Amplification, Southern Blot, Western Blot, Marker, Transduction, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay

    Transformation strategy. A. The original Kozak-like motif in the human TYMS cDNA was modified to the Kozak consensus sequence using partially complementary PCR primers (see Materials and methods ). B. The Kozak-modified TYMS cDNA, TSCD3, was amplified by PCR and subcloned into the cDNA expression vector, pTRE2hyg. C. pTRE2hyg-TS3 or an empty vector was co-transfected with pTet-ON/OFF vectors into a human colorectal cancer cell line, DLD-1. After serial selections, clones resistant to both HygB and G418 were isolated. TS expression in these clones was then examined by immunoblotting.

    Journal: PLoS ONE

    Article Title: Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

    doi: 10.1371/journal.pone.0123076

    Figure Lengend Snippet: Transformation strategy. A. The original Kozak-like motif in the human TYMS cDNA was modified to the Kozak consensus sequence using partially complementary PCR primers (see Materials and methods ). B. The Kozak-modified TYMS cDNA, TSCD3, was amplified by PCR and subcloned into the cDNA expression vector, pTRE2hyg. C. pTRE2hyg-TS3 or an empty vector was co-transfected with pTet-ON/OFF vectors into a human colorectal cancer cell line, DLD-1. After serial selections, clones resistant to both HygB and G418 were isolated. TS expression in these clones was then examined by immunoblotting.

    Article Snippet: Chemicals Hygromycin B (HygB), G418 and doxycycline (Dox) were purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA).

    Techniques: Transformation Assay, Modification, Sequencing, Polymerase Chain Reaction, Amplification, Expressing, Plasmid Preparation, Transfection, Clone Assay, Isolation

    5-FU sensitivity of TFTS66 cells in vitro . A. Effects of 5-FU on the cell cycle of TFTS66 and parental DLD-1 cells. Exponentially growing cells were treated with 5-FU concentrations indicated and subjected to flowcytometry. Fluorescence histograms are shown. B. The design of the in vitro colony formation assays is shown. Fifty thousand TFTS66 and TFC7 cells per dish were grown under the Dox concentrations indicated and treated with the indicated concentrations of 5-FU for 72 h. At Day 10, colonies were counted. Throughout the experiments, cells were maintained in media containing HygB and G418. Each experiment was triplicated. C. Survival curves of TFTS66 and TCF7 cells exposed to 5-FU. The survival fractions were calculated as a percentage of the untreated ( i . e . 0 μM 5-FU) control, and the mean values are plotted against the 5-FU concentration: rectangle, TFTS66; circle, TFC7. The symbols are shaded according to the Dox concentrations. D. The IC50 value in each group was determined as the 5-FU concentration corresponding to 50% survival in the linearized survival curves. Standard error bars are shown at both ends of the linearized survival curves. E. The obtained IC50 values are plotted as a function of the TS expression level determined by immunoblotting (see Fig 2B ): rectangle, TFTS66; circle, TFC7. The symbols are similarly shaded according to the Dox concentrations.

    Journal: PLoS ONE

    Article Title: Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

    doi: 10.1371/journal.pone.0123076

    Figure Lengend Snippet: 5-FU sensitivity of TFTS66 cells in vitro . A. Effects of 5-FU on the cell cycle of TFTS66 and parental DLD-1 cells. Exponentially growing cells were treated with 5-FU concentrations indicated and subjected to flowcytometry. Fluorescence histograms are shown. B. The design of the in vitro colony formation assays is shown. Fifty thousand TFTS66 and TFC7 cells per dish were grown under the Dox concentrations indicated and treated with the indicated concentrations of 5-FU for 72 h. At Day 10, colonies were counted. Throughout the experiments, cells were maintained in media containing HygB and G418. Each experiment was triplicated. C. Survival curves of TFTS66 and TCF7 cells exposed to 5-FU. The survival fractions were calculated as a percentage of the untreated ( i . e . 0 μM 5-FU) control, and the mean values are plotted against the 5-FU concentration: rectangle, TFTS66; circle, TFC7. The symbols are shaded according to the Dox concentrations. D. The IC50 value in each group was determined as the 5-FU concentration corresponding to 50% survival in the linearized survival curves. Standard error bars are shown at both ends of the linearized survival curves. E. The obtained IC50 values are plotted as a function of the TS expression level determined by immunoblotting (see Fig 2B ): rectangle, TFTS66; circle, TFC7. The symbols are similarly shaded according to the Dox concentrations.

    Article Snippet: Chemicals Hygromycin B (HygB), G418 and doxycycline (Dox) were purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA).

    Techniques: In Vitro, Fluorescence, Concentration Assay, Expressing

    Mitochondrial disruption specifically induces the ESRE network. (A) Expression of ESRE-containing genes in worms exposed to 50 μM rotenone or 50 μM antimycin A. Fold changes are normalized to vehicle (DMSO) control. (B) Expression of ESRE-containing and non-ESRE containing genes (red label) after treatment with 1 mM 1,10-phenanthroline (Phe) or with 80 μg/mL hygromycin B (Hygro). irg-1 is a hygromycin-responsive gene used as a control. Fold changes are normalized to vehicle (DMSO) control. Error bars represent SEM.

    Journal: PLoS Genetics

    Article Title: A conserved mitochondrial surveillance pathway is required for defense against Pseudomonas aeruginosa

    doi: 10.1371/journal.pgen.1006876

    Figure Lengend Snippet: Mitochondrial disruption specifically induces the ESRE network. (A) Expression of ESRE-containing genes in worms exposed to 50 μM rotenone or 50 μM antimycin A. Fold changes are normalized to vehicle (DMSO) control. (B) Expression of ESRE-containing and non-ESRE containing genes (red label) after treatment with 1 mM 1,10-phenanthroline (Phe) or with 80 μg/mL hygromycin B (Hygro). irg-1 is a hygromycin-responsive gene used as a control. Fold changes are normalized to vehicle (DMSO) control. Error bars represent SEM.

    Article Snippet: Prior to exposure to small molecules, synchronized young adult worms were washed from NGM plates seeded with OP50, and resuspended in S basal supplemented with 50 μM antimycin A (Sigma), 50 μM rotenone (Sigma), 80 μg/mL hygromycin B, 60 μM tunicamycin (TOCRIS Bioscience), or 12.5 μM bortezomib (LC Laboratories) in the presence of OP50.

    Techniques: Expressing

    Characterization of blasticidin S and hygromycin B resistances in B. burgdorferi . (A) Maps of shuttle vectors pBSV2B and pBSV2H. IR, inverted repeats; cp9 ori , origin or replication of B. burgdorferi plasmid cp9; colE1 ori , E. coli origin of replication; MCS, multicloning site; arr-2 , rifampin resistance gene for selection in E. coli ; P flgB , B. burgdorferi flagellar rod operon promoter; bsd Bb , B. burgdorferi codon-optimized blasticidin S deaminase-encoding gene; hph Bb , B. burgdorferi codon-optimized hygromycin B phosphotransferase-encoding gene. The maps are not drawn to scale. (B) Plate map showing the final antibiotic concentrations used for cross-resistance testing. Each concentration was tested in two adjacent wells. Concentrations routinely used for selection are indicated by the arrow. (C) Schematic representation of color change of the growth medium from red (absence of spirochete growth) to orange/yellow (presence of spirochete growth). A line marks the boundary between growth and no growth in an antibiotic concentration series. The lowest antibiotic concentration that blocked growth was identified as the MIC. (D to H) Susceptibility test of each resistance-carrying strain to various antibiotic concentrations according to the plate layout shown in panel B. The plates were incubated to allow for growth-dependent acidification of the medium and change in phenol red pH indicator color from red to orange and yellow, as depicted in panel C. Images were obtained using colorimetric imaging of the individual plates. MIC boundaries are marked by dark lines, and the MIC values are summarized in Table 3 . The strains used are listed above each image.

    Journal: Applied and Environmental Microbiology

    Article Title: Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease Spirochete Borrelia burgdorferi

    doi: 10.1128/AEM.01824-18

    Figure Lengend Snippet: Characterization of blasticidin S and hygromycin B resistances in B. burgdorferi . (A) Maps of shuttle vectors pBSV2B and pBSV2H. IR, inverted repeats; cp9 ori , origin or replication of B. burgdorferi plasmid cp9; colE1 ori , E. coli origin of replication; MCS, multicloning site; arr-2 , rifampin resistance gene for selection in E. coli ; P flgB , B. burgdorferi flagellar rod operon promoter; bsd Bb , B. burgdorferi codon-optimized blasticidin S deaminase-encoding gene; hph Bb , B. burgdorferi codon-optimized hygromycin B phosphotransferase-encoding gene. The maps are not drawn to scale. (B) Plate map showing the final antibiotic concentrations used for cross-resistance testing. Each concentration was tested in two adjacent wells. Concentrations routinely used for selection are indicated by the arrow. (C) Schematic representation of color change of the growth medium from red (absence of spirochete growth) to orange/yellow (presence of spirochete growth). A line marks the boundary between growth and no growth in an antibiotic concentration series. The lowest antibiotic concentration that blocked growth was identified as the MIC. (D to H) Susceptibility test of each resistance-carrying strain to various antibiotic concentrations according to the plate layout shown in panel B. The plates were incubated to allow for growth-dependent acidification of the medium and change in phenol red pH indicator color from red to orange and yellow, as depicted in panel C. Images were obtained using colorimetric imaging of the individual plates. MIC boundaries are marked by dark lines, and the MIC values are summarized in Table 3 . The strains used are listed above each image.

    Article Snippet: Ampicillin was purchased from Fisher Scientific, blasticidin S and hygromycin B were from Invivogen, and all other antibiotics and biliverdin hydrochloride were from Sigma-Aldrich.

    Techniques: Plasmid Preparation, Selection, Concentration Assay, Incubation, Imaging

    pERα-IRES MDA-MB-231 transfectants: ERα expression via Western immunoblot analysis. A. MDA-MB-231 clones selected for Hygromycin B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-231 parental cell-line represented the negative control. B. Representation of ERα steady state expression values. The values of ERα expression were normalized to tubulin expression, with MCF7 value being one unit.

    Journal: PLoS ONE

    Article Title: ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

    doi: 10.1371/journal.pone.0031977

    Figure Lengend Snippet: pERα-IRES MDA-MB-231 transfectants: ERα expression via Western immunoblot analysis. A. MDA-MB-231 clones selected for Hygromycin B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-231 parental cell-line represented the negative control. B. Representation of ERα steady state expression values. The values of ERα expression were normalized to tubulin expression, with MCF7 value being one unit.

    Article Snippet: Both cell lines were transfected with the pERα-IRES construct as before and selection with hygromycin B was carried out under minimal estrogen exposure conditions, as previously outlined.

    Techniques: Multiple Displacement Amplification, Expressing, Western Blot, Clone Assay, Positive Control, Negative Control

    pERα-IRES MDA-MB-435 transfectants: Western immunoblot analysis of ERα protein. A. MDA-MB-435 cell clones selected for Hygromycin B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-435 parental cell-line represented the negative control. B. Representation of ERα steady state expression values. The values of ERα expression were normalized to α tubulin expression in the cells.

    Journal: PLoS ONE

    Article Title: ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

    doi: 10.1371/journal.pone.0031977

    Figure Lengend Snippet: pERα-IRES MDA-MB-435 transfectants: Western immunoblot analysis of ERα protein. A. MDA-MB-435 cell clones selected for Hygromycin B resistance were lysed and ERα expression was tested by Western immunoblot analysis. ERα positive MCF-7 cell line was used as a positive control. MDA-MB-435 parental cell-line represented the negative control. B. Representation of ERα steady state expression values. The values of ERα expression were normalized to α tubulin expression in the cells.

    Article Snippet: Both cell lines were transfected with the pERα-IRES construct as before and selection with hygromycin B was carried out under minimal estrogen exposure conditions, as previously outlined.

    Techniques: Multiple Displacement Amplification, Western Blot, Clone Assay, Expressing, Positive Control, Negative Control

    Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.

    Journal: PLoS ONE

    Article Title: ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

    doi: 10.1371/journal.pone.0031977

    Figure Lengend Snippet: Characterization of ectopically expressed RNAs by long range RT-PCR. MDA-MB-231 parental cell line (231-parental), its pcDNA3-ERα stable transfectant (ERα-2), and its ERα-IRES stable transfectants (ERα-IRES-5 and ERα-IRES-3) were analyzed for expression of ERα–harboring transcript (1.8 kb), Hygromycin B resistance gene-containing transcript (1.0 kb), and ERα-IRES-Hygro R fused transcript (3.2 kb), by RT followed by long range PCR amplification. pERα-IRES DNA served as a PCR positive control for the ERα cDNA primers (1.8 kb), the Hygromycin B resistance gene ORF primers (1.0 kb), and the 5′ sense ERα primer plus 3′ antisense Hygro R fused ORFs primers (3.5 kb). A First four lanes from left contain the ERα cDNA primers; lanes 5–8 the 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. B. The 5′ sense ERα primer together with the 3′ antisense Hygro R gene primer. C. Lanes 1 and 2 from left, the ERα primers. Lanes 3 and 4 the Hygro R gene primers. Primer sequences are detailed in the “ Methods ” section.

    Article Snippet: Both cell lines were transfected with the pERα-IRES construct as before and selection with hygromycin B was carried out under minimal estrogen exposure conditions, as previously outlined.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Transfection, Expressing, Polymerase Chain Reaction, Amplification, Positive Control

    Southern blot evidence for pMAD10-mediated  URA5 Hc  gene disruption in strain G184AS. Genomic DNAs from transformation recipient strain G184AS (lanes 1) and the four hygromycin B-resistant, uracil-auxotrophic transformants (lanes 2 to 5) were digested with  Cla I, electrophoresed, Southern blotted, and probed with radiolabeled fragments of the  URA5 Hc  gene, the 0.4-kb internal  URA5 Hc  fragment deleted from pMAD10, and the inserted selectable marker  hph . The four uracil auxotrophs (lanes 2 to 5) have lost the native genomic band that hybridizes with the  URA5 Hc  probe and gained a new, larger band due to  hph  insertion that hybridizes with both the  URA5 Hc  and  hph  probes, as well as failing to show any hybridization with the 0.4-kb fragment deleted from the disruption construct pMAD10. Fragment sizes were estimated by comparison to molecular size standards in the ethidium bromide-stained gel.

    Journal: Journal of Bacteriology

    Article Title: Rare Homologous Gene Targeting in Histoplasma capsulatum: Disruption of the URA5Hc Gene by Allelic Replacement

    doi:

    Figure Lengend Snippet: Southern blot evidence for pMAD10-mediated URA5 Hc gene disruption in strain G184AS. Genomic DNAs from transformation recipient strain G184AS (lanes 1) and the four hygromycin B-resistant, uracil-auxotrophic transformants (lanes 2 to 5) were digested with Cla I, electrophoresed, Southern blotted, and probed with radiolabeled fragments of the URA5 Hc gene, the 0.4-kb internal URA5 Hc fragment deleted from pMAD10, and the inserted selectable marker hph . The four uracil auxotrophs (lanes 2 to 5) have lost the native genomic band that hybridizes with the URA5 Hc probe and gained a new, larger band due to hph insertion that hybridizes with both the URA5 Hc and hph probes, as well as failing to show any hybridization with the 0.4-kb fragment deleted from the disruption construct pMAD10. Fragment sizes were estimated by comparison to molecular size standards in the ethidium bromide-stained gel.

    Article Snippet: Interestingly, in addition to four genuine URA5 Hc gene-disrupted transformants, we obtained a hygromycin B- and FOA-resistant colony that was uracil prototrophic and had no genomic alteration at the URA5 Hc locus detectable by PCR (Fig. , lanes 6), presumably resulting from spontaneous or transformation-induced mutation to FOA resistance in a yeast transformed to hygromycin B resistance due to ectopic integration or linear plasmid formation by the transforming DNA.

    Techniques: Southern Blot, Transformation Assay, Marker, Hybridization, Construct, Staining

    PCR evidence for pMAD10-mediated  URA5 Hc  gene disruption in strain G184AS. Genomic DNAs from transformation recipient strain G184AS (lanes 1), the four hygromycin B-resistant, uracil-auxotrophic transformants (lanes 2 to 5), and a hygromycin B-resistant, uracil-prototrophic isolate (lanes 6) were subjected to PCR using primers targeted to a  URA5 Hc  region upstream of the site of  hph  insertion (primer set A) or primers targeted to sites that flank the site of  hph  insertion (primer set B). The four uracil auxotrophs (lanes 2 to 5) have lost the native product and gained a new larger product due to  hph  insertion with primer set B. The uracil prototroph (lanes 6) shows both the unchanged native product and the larger product with primer set B, due to ectopic integration or linear plasmid formation by the transforming DNA. Lanes M, molecular size standards (λ/ Hin dIII and φX/ Hae III).

    Journal: Journal of Bacteriology

    Article Title: Rare Homologous Gene Targeting in Histoplasma capsulatum: Disruption of the URA5Hc Gene by Allelic Replacement

    doi:

    Figure Lengend Snippet: PCR evidence for pMAD10-mediated URA5 Hc gene disruption in strain G184AS. Genomic DNAs from transformation recipient strain G184AS (lanes 1), the four hygromycin B-resistant, uracil-auxotrophic transformants (lanes 2 to 5), and a hygromycin B-resistant, uracil-prototrophic isolate (lanes 6) were subjected to PCR using primers targeted to a URA5 Hc region upstream of the site of hph insertion (primer set A) or primers targeted to sites that flank the site of hph insertion (primer set B). The four uracil auxotrophs (lanes 2 to 5) have lost the native product and gained a new larger product due to hph insertion with primer set B. The uracil prototroph (lanes 6) shows both the unchanged native product and the larger product with primer set B, due to ectopic integration or linear plasmid formation by the transforming DNA. Lanes M, molecular size standards (λ/ Hin dIII and φX/ Hae III).

    Article Snippet: Interestingly, in addition to four genuine URA5 Hc gene-disrupted transformants, we obtained a hygromycin B- and FOA-resistant colony that was uracil prototrophic and had no genomic alteration at the URA5 Hc locus detectable by PCR (Fig. , lanes 6), presumably resulting from spontaneous or transformation-induced mutation to FOA resistance in a yeast transformed to hygromycin B resistance due to ectopic integration or linear plasmid formation by the transforming DNA.

    Techniques: Polymerase Chain Reaction, Transformation Assay, Plasmid Preparation

    Expression plasmids for cellulase production. Schematic presentation of the two cellulase expression plasmids used in this study. Both plasmids contain the hygromycin B expression cassette as fungal selection marker followed by either the tef1 or the cDNA1 promoter region and the coding and terminator region of the respective cellulase gene (e.g., the endoglucanase encoding gene cel7b )

    Journal: Applied Microbiology and Biotechnology

    Article Title: A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background

    doi: 10.1007/s00253-011-3674-8

    Figure Lengend Snippet: Expression plasmids for cellulase production. Schematic presentation of the two cellulase expression plasmids used in this study. Both plasmids contain the hygromycin B expression cassette as fungal selection marker followed by either the tef1 or the cDNA1 promoter region and the coding and terminator region of the respective cellulase gene (e.g., the endoglucanase encoding gene cel7b )

    Article Snippet: Selection media contained 50 μg/mL hygromycin B (Roth).

    Techniques: Expressing, Selection, Marker

    A representative colony at 7 days post-hygromycin B selection Phase contrast, 4× magnification. Scale bar represents 1,000 μm.

    Journal: Bio-protocol

    Article Title: Quantitative Live-cell Reporter Assay for Noncanonical Wnt Activity

    doi: 10.21769/BioProtoc.2762

    Figure Lengend Snippet: A representative colony at 7 days post-hygromycin B selection Phase contrast, 4× magnification. Scale bar represents 1,000 μm.

    Article Snippet: 6-well plate 48-well tissue culture plate (Corning, Costar® , catalog number: 3548) 5 ml round-bottom tubes with 35 μm cell strainer snap cap (Corning, Falcon® , catalog number: 352235) NIH/3T3 Flp-In cells (Thermo Fisher Scientific, Invitrogen™, catalog number: R76107) pCAG-GFP (available upon request), or any GFP plasmid suitable for mammalian expression pEF5-FRT-GFP-Kif26b (Addgene, catalog number: 102862) reporter construct pOG44 Flp-Recombinase expression vector (Thermo Fisher Scientific, Invitrogen™, catalog number: V600520) Recombinant Wnt5a (R & D Systems, catalog number: 654-WN-010) Genjet In Vitro Transfection Reagent for NIH/3T3 cells (SignaGen Laboratories, catalog number: SL100488, 3T3) Hygromycin B (50 mg/ml solution) (Corning, Mediatech, catalog number: 30-240-CR) Poly-D-lysine (Sigma-Aldrich, catalog number: P6407-10X5MG) Wnt-C59 (Cellagen Technology, catalog number: C7641-2s) Trypsin EDTA (Corning, Mediatech, catalog number: 25-052-CI) Dulbecco's modified Eagle's medium (DMEM) (Corning, Mediatech, catalog number: 15-017-CV) Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco™, catalog number: 16000069) Note: The FBS is used directly without heat-inactivation.

    Techniques: Selection

    Schematic illustration of the promoter region and the methylation frequencies within the 35S promoter. A , Within the transfer DNA (T-DNA) the resistance marker ( hpt II) is driven by a  nopaline synthase  (NOS) promoter and confers resistance to the antibiotic hygromycin B. The gene of interest (GOI) is driven by a cauliflower mosaic virus promoter (35S). For methylation analysis a 294 bp fragment of the NOS promoter or a 346 bp fragment of the 35S promoter were amplified with the indicated bisulfite primers. Positions of the restriction sites of  Xba I and  Eco RV are indicated.  B , Graphical output of the 35S promoter wide methylation in two consecutive generations of line PNA 1.2 (T 2  and T 3  stage). DNA was isolated from seedlings 15 days post germination (dpg). Filled symbols indicate cytosine methylation, whereas empty symbols indicate a lack of methylation. Red circles represent CG sites, blue squares represent CHG sites and green triangles represent CHH sites, whereas H = A,T or C. Analysis was performed with CyMATE [  127 ]. The upper part illustrates the spatial distribution of the methylation sites within the promoter sequence. Mean cytosine methylation was calculated for the three methylation sites (CG, CHG and CHH) from individually picked clones (±SEM, n = 12 clones).

    Journal: BMC Plant Biology

    Article Title: Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants

    doi: 10.1186/1471-2229-13-99

    Figure Lengend Snippet: Schematic illustration of the promoter region and the methylation frequencies within the 35S promoter. A , Within the transfer DNA (T-DNA) the resistance marker ( hpt II) is driven by a nopaline synthase (NOS) promoter and confers resistance to the antibiotic hygromycin B. The gene of interest (GOI) is driven by a cauliflower mosaic virus promoter (35S). For methylation analysis a 294 bp fragment of the NOS promoter or a 346 bp fragment of the 35S promoter were amplified with the indicated bisulfite primers. Positions of the restriction sites of Xba I and Eco RV are indicated. B , Graphical output of the 35S promoter wide methylation in two consecutive generations of line PNA 1.2 (T 2 and T 3 stage). DNA was isolated from seedlings 15 days post germination (dpg). Filled symbols indicate cytosine methylation, whereas empty symbols indicate a lack of methylation. Red circles represent CG sites, blue squares represent CHG sites and green triangles represent CHH sites, whereas H = A,T or C. Analysis was performed with CyMATE [ 127 ]. The upper part illustrates the spatial distribution of the methylation sites within the promoter sequence. Mean cytosine methylation was calculated for the three methylation sites (CG, CHG and CHH) from individually picked clones (±SEM, n = 12 clones).

    Article Snippet: The hybrid offspring (hemizygous to the transgene) should be theoretically fully resistant to hygromycin B.

    Techniques: Methylation, Marker, Amplification, Isolation, Sequencing, Clone Assay

    Progressive loss of transgene expression in subsequent generations. A , Gene expression analysis in homozygous plants of 5 independent N. attenuata lines, comparing two consecutive generations (T 2 and T 3 ). Transcript abundance of the ICE and PNA transgenes was determined by qRT-PCR on cDNA of rosette-stage leaves (30 dpg). Bars indicate the Δ C T mean expression (log 2 fold expression) relative to actin as the reference gene (±SD, n = 3 plants). The T 3 generation of the lines ICE 4.4.1, PNA 1.2.1 and PNA 10.1.1 indicated a strong reduction of transgene expression. Lines ICE 1.1.1 and PNA 8.6.1 showed stable and high transgene expression in the T 3 generation. B , Photographs of 15-day-old T 3 seedlings grown on a hygromycin B containing GB5 medium. Sensitivity to hygromycin was indicated by small, yellowish seedlings and the lack of root hairs. Resistant seedlings were larger, with dark green cotyledons and clearly developed root hairs. The lines ICE 4.4.1, PNA 1.2.1 and PNA 10.1.1 showed sensitivity to hygromycin in the T 3 generation similar to wild-type, whereas the lines ICE 1.1.1 and PNA 8.6.1 remained hygromycin resistance. For each line seeds of three different plants were analyzed and wild-type seeds used as negative control.

    Journal: BMC Plant Biology

    Article Title: Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants

    doi: 10.1186/1471-2229-13-99

    Figure Lengend Snippet: Progressive loss of transgene expression in subsequent generations. A , Gene expression analysis in homozygous plants of 5 independent N. attenuata lines, comparing two consecutive generations (T 2 and T 3 ). Transcript abundance of the ICE and PNA transgenes was determined by qRT-PCR on cDNA of rosette-stage leaves (30 dpg). Bars indicate the Δ C T mean expression (log 2 fold expression) relative to actin as the reference gene (±SD, n = 3 plants). The T 3 generation of the lines ICE 4.4.1, PNA 1.2.1 and PNA 10.1.1 indicated a strong reduction of transgene expression. Lines ICE 1.1.1 and PNA 8.6.1 showed stable and high transgene expression in the T 3 generation. B , Photographs of 15-day-old T 3 seedlings grown on a hygromycin B containing GB5 medium. Sensitivity to hygromycin was indicated by small, yellowish seedlings and the lack of root hairs. Resistant seedlings were larger, with dark green cotyledons and clearly developed root hairs. The lines ICE 4.4.1, PNA 1.2.1 and PNA 10.1.1 showed sensitivity to hygromycin in the T 3 generation similar to wild-type, whereas the lines ICE 1.1.1 and PNA 8.6.1 remained hygromycin resistance. For each line seeds of three different plants were analyzed and wild-type seeds used as negative control.

    Article Snippet: The hybrid offspring (hemizygous to the transgene) should be theoretically fully resistant to hygromycin B.

    Techniques: Expressing, Quantitative RT-PCR, Negative Control

    Segregation analysis of resistance marker loci in Nicotiana attenuata. Histogram of the sensitivity rates of 113 independently transformed N. attenuata lines due to segregation of the resistance marker gene monitored over three generations (T 1 –T 3 ), overlaid with smoothed density plots. Seedlings with a sensitivity rate between 10–50% were considered to descend from a hemizygous plant. More than 50% sensitivity was interpreted as gene silencing of the hygromycin B resistance marker and a sensitivity rate around 6.25% indicated two independent segregating loci. A , Sensitivity rates of T 1 seedlings collected from 113 independently transformed T 0 plant lines. Seedlings with the desired sensitivity rate between 10–50% were chosen for further inbreeding (indicated in black). B , Sensitivity rates of T 2 seedlings collected from 951 T 1 plants. The offspring of nine to ten plants were analyzed per plant line. Descendants from homozygous plants (0% sensitivity) were chosen for further inbreeding (indicated in black). C , Sensitivity rates of T 3 seedlings collected from 149 T 2 plants homozygous to the transgene. Any occurring sensitivity to the resistance marker was considered as gene silencing. Desired plant lines with sustained resistance were indicated in black.

    Journal: BMC Plant Biology

    Article Title: Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants

    doi: 10.1186/1471-2229-13-99

    Figure Lengend Snippet: Segregation analysis of resistance marker loci in Nicotiana attenuata. Histogram of the sensitivity rates of 113 independently transformed N. attenuata lines due to segregation of the resistance marker gene monitored over three generations (T 1 –T 3 ), overlaid with smoothed density plots. Seedlings with a sensitivity rate between 10–50% were considered to descend from a hemizygous plant. More than 50% sensitivity was interpreted as gene silencing of the hygromycin B resistance marker and a sensitivity rate around 6.25% indicated two independent segregating loci. A , Sensitivity rates of T 1 seedlings collected from 113 independently transformed T 0 plant lines. Seedlings with the desired sensitivity rate between 10–50% were chosen for further inbreeding (indicated in black). B , Sensitivity rates of T 2 seedlings collected from 951 T 1 plants. The offspring of nine to ten plants were analyzed per plant line. Descendants from homozygous plants (0% sensitivity) were chosen for further inbreeding (indicated in black). C , Sensitivity rates of T 3 seedlings collected from 149 T 2 plants homozygous to the transgene. Any occurring sensitivity to the resistance marker was considered as gene silencing. Desired plant lines with sustained resistance were indicated in black.

    Article Snippet: The hybrid offspring (hemizygous to the transgene) should be theoretically fully resistant to hygromycin B.

    Techniques: Marker, Transformation Assay

    Assaying interactions and relative fitness between kanMX4 and hphEC6 selection markers. a Outline of linearized constructs used in interaction assays. Plasmid elements are not drawn to scale. b Tolerance of yeast strains to combinations of G418 sulfate and hygromycin B. Strains were pre-cultured overnight in 3 mL YM broth with either 200 mg/L G418 disulfate (TLSC001), 200 mg/L hygromycin B (TLSC009), or 200 mg/L of both selection agents (TLSC008 and TLSC010). Pre-cultures were diluted to OD 600 0.1 in fresh YM broth without any selection agent. 2 μL cell suspension of each strain was spotted on solid YM medium with the indicated concentration of G418 disulfate and hygromycin B. Plates were incubated for 2 days at 30 °C and then photographed. c Comparative growth dynamics in MMD broth containing both G418 disulfate and hygromycin B. Error bars indicate one standard deviation. d Fitness equivalence of co-cultivated S. cerevisiae Δput1 strains carrying either the kanMX4 or hphEC6 marker under non-selective conditions. Error bars indicate one standard deviation

    Journal: 3 Biotech

    Article Title: Development of a yeast heterologous expression cassette based on the promoter and terminator elements of the Eremothecium cymbalariae translational elongation factor 1α (EcTEF1) gene

    doi: 10.1007/s13205-018-1224-0

    Figure Lengend Snippet: Assaying interactions and relative fitness between kanMX4 and hphEC6 selection markers. a Outline of linearized constructs used in interaction assays. Plasmid elements are not drawn to scale. b Tolerance of yeast strains to combinations of G418 sulfate and hygromycin B. Strains were pre-cultured overnight in 3 mL YM broth with either 200 mg/L G418 disulfate (TLSC001), 200 mg/L hygromycin B (TLSC009), or 200 mg/L of both selection agents (TLSC008 and TLSC010). Pre-cultures were diluted to OD 600 0.1 in fresh YM broth without any selection agent. 2 μL cell suspension of each strain was spotted on solid YM medium with the indicated concentration of G418 disulfate and hygromycin B. Plates were incubated for 2 days at 30 °C and then photographed. c Comparative growth dynamics in MMD broth containing both G418 disulfate and hygromycin B. Error bars indicate one standard deviation. d Fitness equivalence of co-cultivated S. cerevisiae Δput1 strains carrying either the kanMX4 or hphEC6 marker under non-selective conditions. Error bars indicate one standard deviation

    Article Snippet: The selection agents G418 disulfate and hygromycin B were purchased from Formedium Ltd (Norfolk, UK).

    Techniques: Selection, Construct, Plasmid Preparation, Cell Culture, Concentration Assay, Incubation, Standard Deviation, Marker

    Hygromycin B tolerance of hphEC variants. Strains were pre-cultured overnight in 3 mL YM broth with 200 mg/L G418 disulfate and then diluted to OD 600 0.1 in fresh YM broth without any selection agent. 2 μL cell suspension of each strain was spotted on solid YM medium with the indicated concentration of hygromycin B. Plates were incubated for 2 days at 30 °C and then photographed

    Journal: 3 Biotech

    Article Title: Development of a yeast heterologous expression cassette based on the promoter and terminator elements of the Eremothecium cymbalariae translational elongation factor 1α (EcTEF1) gene

    doi: 10.1007/s13205-018-1224-0

    Figure Lengend Snippet: Hygromycin B tolerance of hphEC variants. Strains were pre-cultured overnight in 3 mL YM broth with 200 mg/L G418 disulfate and then diluted to OD 600 0.1 in fresh YM broth without any selection agent. 2 μL cell suspension of each strain was spotted on solid YM medium with the indicated concentration of hygromycin B. Plates were incubated for 2 days at 30 °C and then photographed

    Article Snippet: The selection agents G418 disulfate and hygromycin B were purchased from Formedium Ltd (Norfolk, UK).

    Techniques: Cell Culture, Selection, Concentration Assay, Incubation

    Concentration of G418 and hygromycin B for the selection of transfected cells. In 24-well plates, 201B7 cells were cultured to confluency and G418 or hygromycin B was added. The cells were examined under the microscope 7 days after the addition of the

    Journal: Tissue Engineering. Part A

    Article Title: Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

    doi: 10.1089/ten.tea.2014.0132

    Figure Lengend Snippet: Concentration of G418 and hygromycin B for the selection of transfected cells. In 24-well plates, 201B7 cells were cultured to confluency and G418 or hygromycin B was added. The cells were examined under the microscope 7 days after the addition of the

    Article Snippet: To determine the conditions for selection, G418 (Wako Pure Chemicals, Osaka, Japan) and/or hygromycin B (Wako Pure Chemicals) were added to the medium and cells were monitored under a microscope after 7 days.

    Techniques: Concentration Assay, Selection, Transfection, Cell Culture, Microscopy

    Time-course of transfected cells. In a six-well plate, 201B7 cells were transfected with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg using suspended method with Lipofectamine LTX or FuGENE HD at 4.5×10 5 cells/well. G418 and hygromycin B were added 4–6

    Journal: Tissue Engineering. Part A

    Article Title: Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

    doi: 10.1089/ten.tea.2014.0132

    Figure Lengend Snippet: Time-course of transfected cells. In a six-well plate, 201B7 cells were transfected with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg using suspended method with Lipofectamine LTX or FuGENE HD at 4.5×10 5 cells/well. G418 and hygromycin B were added 4–6

    Article Snippet: To determine the conditions for selection, G418 (Wako Pure Chemicals, Osaka, Japan) and/or hygromycin B (Wako Pure Chemicals) were added to the medium and cells were monitored under a microscope after 7 days.

    Techniques: Transfection

    Percentage of EGFP and CherryPicker cells over time. Photos of the same experiments in were analyzed to determine the incubation period with G418 and hygromycin B to obtain the cells 100% expressing the reporter genes. Using ImageJ 1.42q regions

    Journal: Tissue Engineering. Part A

    Article Title: Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

    doi: 10.1089/ten.tea.2014.0132

    Figure Lengend Snippet: Percentage of EGFP and CherryPicker cells over time. Photos of the same experiments in were analyzed to determine the incubation period with G418 and hygromycin B to obtain the cells 100% expressing the reporter genes. Using ImageJ 1.42q regions

    Article Snippet: To determine the conditions for selection, G418 (Wako Pure Chemicals, Osaka, Japan) and/or hygromycin B (Wako Pure Chemicals) were added to the medium and cells were monitored under a microscope after 7 days.

    Techniques: Incubation, Expressing

    Translational inhibition abolishes the OCTR-1-controlled innate immune responses. ( A ) qRT-PCR of  abu-1 ,  abu-7 ,  abu-8 ,  abu-12 ,  abu-13 ,  abu-14 , and  abu-15  expression in wild-type animals with or without exposure to G418 and in  octr-1 ( ok371 ) animals exposed to G418.  n  = 3; bar graphs correspond to mean ± SEM.  t -tests were performed between N2 + G418 and  octr-1  + G418. *indicates significant difference. ( B ) qRT-PCR of  abu-1 ,  abu-7 ,  abu-8 ,  abu-12 ,  abu-13 ,  abu-14 , and  abu-15  expression in wild-type animals with or without exposure to Hygrocymin B and in  octr-1 ( ok371 ) animals exposed to Hygromycin B.  n  = 3; bar graphs correspond to mean ± SEM.  t -tests were performed between N2 + G418 and  octr-1  + G418. *indicates significant difference. ( C ) Wild-type and  octr-1 ( ok371 ) animals were exposed to NGM/ E. coli  OP50 (control) or NGM/ E. coli  OP50 containing G418 or Hygromycin B, and scored for survival over time. WT + No inhibitor versus  octr-1 ( ok371 )+No inhibitor:  p  = 0.7521; WT + G418 versus  octr-1 ( ok371 )+G418:  p

    Journal: Scientific Reports

    Article Title: Neuronal GPCR OCTR-1 regulates innate immunity by controlling protein synthesis in Caenorhabditis elegans

    doi: 10.1038/srep36832

    Figure Lengend Snippet: Translational inhibition abolishes the OCTR-1-controlled innate immune responses. ( A ) qRT-PCR of abu-1 , abu-7 , abu-8 , abu-12 , abu-13 , abu-14 , and abu-15 expression in wild-type animals with or without exposure to G418 and in octr-1 ( ok371 ) animals exposed to G418. n  = 3; bar graphs correspond to mean ± SEM. t -tests were performed between N2 + G418 and octr-1  + G418. *indicates significant difference. ( B ) qRT-PCR of abu-1 , abu-7 , abu-8 , abu-12 , abu-13 , abu-14 , and abu-15 expression in wild-type animals with or without exposure to Hygrocymin B and in octr-1 ( ok371 ) animals exposed to Hygromycin B. n  = 3; bar graphs correspond to mean ± SEM. t -tests were performed between N2 + G418 and octr-1  + G418. *indicates significant difference. ( C ) Wild-type and octr-1 ( ok371 ) animals were exposed to NGM/ E. coli OP50 (control) or NGM/ E. coli OP50 containing G418 or Hygromycin B, and scored for survival over time. WT + No inhibitor versus octr-1 ( ok371 )+No inhibitor: p  = 0.7521; WT + G418 versus octr-1 ( ok371 )+G418: p

    Article Snippet: Translation inhibitor treatment Three thousand one hundred twenty five μl of 50 mg/ml G418 (Roche, New York, NY) or 750 μl of 50 mg/ml Hygromycin B (Mediatech, Inc. Manassas, VA) was added to 250 ml NGM medium, respectively, to make corresponding G418 or Hygromycin B plates using 6 cm Petri dishes.

    Techniques: Inhibition, Quantitative RT-PCR, Expressing

    NAT as a selectable marker that is compatible with paromomycin and/or hygromycin B resistant genes. a Differential interference contrast (DIC) images showing wild type cells, ift54 mutant and rescued cells. Please note that ift54 did not have flagella and flagellar formation was rescued in IFT54- HA expressing cells. Bar, 10 μm. b A diagram showing construct that harbors expression cassettes of IFT54 -HA and NAT . c Expression of IFT54- HA in strains harboring paromomycin resistant gene by using NAT as a selectable marker. The construct listed above was transformed into ift54, which harbors paromomycin resistant gene . The transformants were selected on agar plates supplemented with 10 µg/ml NTC. Cell lysates from randomly picked transformants were subjected to immunoblotting using the indicated antibodies. CDPK3 was used as a loading control. *Denotes non-specific bands. Wild type (WT) and ift54 cells were used as control. d Expression of IFT54- HA in strains harboring paromomycin and hygromycin B resistant genes by using NAT as a selectable marker. wdr92 ::WDR92-YFP strains with both paromomycin and hygromycin B resistant genes were transformed. The expression of IFT54- HA from cells grown on selection plate was examined by immunoblotting. WT and wdr92 ::WDR92-YFP (paro/hygro) cells were used as control

    Journal: Plant Methods

    Article Title: Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas

    doi: 10.1186/s13007-019-0526-5

    Figure Lengend Snippet: NAT as a selectable marker that is compatible with paromomycin and/or hygromycin B resistant genes. a Differential interference contrast (DIC) images showing wild type cells, ift54 mutant and rescued cells. Please note that ift54 did not have flagella and flagellar formation was rescued in IFT54- HA expressing cells. Bar, 10 μm. b A diagram showing construct that harbors expression cassettes of IFT54 -HA and NAT . c Expression of IFT54- HA in strains harboring paromomycin resistant gene by using NAT as a selectable marker. The construct listed above was transformed into ift54, which harbors paromomycin resistant gene . The transformants were selected on agar plates supplemented with 10 µg/ml NTC. Cell lysates from randomly picked transformants were subjected to immunoblotting using the indicated antibodies. CDPK3 was used as a loading control. *Denotes non-specific bands. Wild type (WT) and ift54 cells were used as control. d Expression of IFT54- HA in strains harboring paromomycin and hygromycin B resistant genes by using NAT as a selectable marker. wdr92 ::WDR92-YFP strains with both paromomycin and hygromycin B resistant genes were transformed. The expression of IFT54- HA from cells grown on selection plate was examined by immunoblotting. WT and wdr92 ::WDR92-YFP (paro/hygro) cells were used as control

    Article Snippet: Reagents Paromomycin and hygromycin B were purchased from Merck Millipore, USA, while NTC was obtained from Jena biosciences, Germany.

    Techniques: Marker, Mutagenesis, Expressing, Construct, Transformation Assay, Selection

    No cross-resistance to NTC in strains with paromomycin and/or hygromycin B resistance. Wild type (WT) cells and transgenic strains with resistance to paromomycin, hygromycin B or both were grown for 4 days on TAP agar plates without antibiotics ( a ) or supplemented with paromomycin ( b ), hygromycin B ( c ) or NTC ( d ). paro, paromomycin resistant strains; hygro, hygromycin B resistant strains; paro/hygro, paromomycin and hygromycin B double resistance strains. Data shown are representative of three experiments

    Journal: Plant Methods

    Article Title: Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas

    doi: 10.1186/s13007-019-0526-5

    Figure Lengend Snippet: No cross-resistance to NTC in strains with paromomycin and/or hygromycin B resistance. Wild type (WT) cells and transgenic strains with resistance to paromomycin, hygromycin B or both were grown for 4 days on TAP agar plates without antibiotics ( a ) or supplemented with paromomycin ( b ), hygromycin B ( c ) or NTC ( d ). paro, paromomycin resistant strains; hygro, hygromycin B resistant strains; paro/hygro, paromomycin and hygromycin B double resistance strains. Data shown are representative of three experiments

    Article Snippet: Reagents Paromomycin and hygromycin B were purchased from Merck Millipore, USA, while NTC was obtained from Jena biosciences, Germany.

    Techniques: Transgenic Assay

    HA-Rom2(733-1199) induces a different growth phenotype than the full-length protein. (A) In a series of 10-fold dilutions derived from a starting suspension of 5 × 10 6 conidia ml −1 of the indicated strains, aliquots of 3 μl were spotted onto AMM supplemented with the indicated amount of doxycycline (Doxy) and incubated for 48 h at 37°C. AMM agar was additionally supplemented with hygromycin B to avoid loss of the respective construct. (B) A total of 5 × 10 3 conidia ml −1 of the indicated strains were inoculated in the wells on plates in liquid AMM supplemented with no doxycycline (top panels) or 5 μg ml −1 doxycycline (bottom panels). The plates were incubated at 37°C for 16 h. Bar, 250 μm.

    Journal: Eukaryotic Cell

    Article Title: Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

    doi: 10.1128/EC.00246-12

    Figure Lengend Snippet: HA-Rom2(733-1199) induces a different growth phenotype than the full-length protein. (A) In a series of 10-fold dilutions derived from a starting suspension of 5 × 10 6 conidia ml −1 of the indicated strains, aliquots of 3 μl were spotted onto AMM supplemented with the indicated amount of doxycycline (Doxy) and incubated for 48 h at 37°C. AMM agar was additionally supplemented with hygromycin B to avoid loss of the respective construct. (B) A total of 5 × 10 3 conidia ml −1 of the indicated strains were inoculated in the wells on plates in liquid AMM supplemented with no doxycycline (top panels) or 5 μg ml −1 doxycycline (bottom panels). The plates were incubated at 37°C for 16 h. Bar, 250 μm.

    Article Snippet: For transformation, AMM agar plates supplemented with 1.2 M sorbitol were used along with 0.5 μg ml−1 doxycycline, 0.1 μg ml−1 pyrithiamine (sc-236525; Santa Cruz Biotechnologies, Santa Cruz, CA), and 200 μg ml−1 hygromycin B (p21-014; PAA Laboratories, Pasching, Austria) as required.

    Techniques: Derivative Assay, Incubation, Construct

    IGR IRES loop 3 mutants bind the 80S ribosomes. ( A ) Assembly of 80S ribosomes on CrPV intergenic region (IGR) IRES loop 3 mutants in rabbit reticulocyte lysate. Radiolabeled CrPV IRES RNAs were incubated in RRL supplemented with hygromycin B for 20 min before separation of initiation complexes on a 15–30% sucrose gradient. Free and 80S-bound IRES complexes are indicated. ( B ) Approximate on- and off-rates of IRES-ribosome binding measured by filter binding. The on-rate experiment measures the association of IRES with ribosomes or ribosomal subunits as a function of time. Pure shrimp ribosomes were used for the on-rate experiment. The off-rate experiment used unlabeled competitor IRES RNA to detect dissociation of IRES from ribosomes as a function of time. Purified yeast subunits were used for the off-rate experiment. DOI: http://dx.doi.org/10.7554/eLife.08146.010

    Journal: eLife

    Article Title: A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

    doi: 10.7554/eLife.08146

    Figure Lengend Snippet: IGR IRES loop 3 mutants bind the 80S ribosomes. ( A ) Assembly of 80S ribosomes on CrPV intergenic region (IGR) IRES loop 3 mutants in rabbit reticulocyte lysate. Radiolabeled CrPV IRES RNAs were incubated in RRL supplemented with hygromycin B for 20 min before separation of initiation complexes on a 15–30% sucrose gradient. Free and 80S-bound IRES complexes are indicated. ( B ) Approximate on- and off-rates of IRES-ribosome binding measured by filter binding. The on-rate experiment measures the association of IRES with ribosomes or ribosomal subunits as a function of time. Pure shrimp ribosomes were used for the on-rate experiment. The off-rate experiment used unlabeled competitor IRES RNA to detect dissociation of IRES from ribosomes as a function of time. Purified yeast subunits were used for the off-rate experiment. DOI: http://dx.doi.org/10.7554/eLife.08146.010

    Article Snippet: Furthermore, we also show the location of the pretranslocation toeprint by using hygromycin B in RRL ( ).

    Techniques: Incubation, Binding Assay, Purification

    New tetracycline- and AID-controlled vectors. (a) Cloning sites of pUHD-AID. Part of the AID tag is shown (the AID tag is 228 amino acid long (~25 kDa)). The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown (except the two BamHI sites). *: termination codon. A puromycin-resistant gene is present in the construct for antibiotic selection in mammalian cells. The complete sequence of pUHD-AID can be found in the Supplemental information. (b) Cloning sites of pRevTRE-AID. The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown. *: termination codon. A hygromycin-resistant gene is expressed from this construct to allow antibiotic selection in mammalian cells. The complete sequence of pRevTRE-AID can be found in the Supplemental information. (c) Workflow for conditional gene inactivation using pUHD-AID or pRevTRE-AID. (1) Silence mutations (changing 3–4 bases) can be introduced into the cDNA to render the cDNA resistant to the CRISPR-Cas9. Alternatively, CRISPR-Cas9 that targets the endogenous gene but not the cDNA (e.g. against a intron-exon boundary) can be used. (2) Cloning sites are shown in panels A and B. (3–4) After cells are transfected with DNA or infected with retroviruses, they are selected with puromycin (pUHD-AID) or hygromycin B (pRevTRE-AID). (5) The cells are then transfected with CRISPR-Cas9 against the gene of interest. Various selection or sorting methods can be used to enrich the transfected cells. (6) Although the entire workflow can be initiated using cells stably expressing TIR1, we found that putting TIR1 into the cells at this stage generated more clones that could effectively respond to IAA. (7) Single clones are isolated and analysed for clones lacking the expression of the endogenous gene of interest, but expressing the AID-tagged version (ideally at a level similar to the endogenous protein) which can be eliminated in the presence of Dox and IAA.

    Journal: Cell Cycle

    Article Title: Conditional gene inactivation by combining tetracycline-mediated transcriptional repression and auxin-inducible degron-mediated degradation

    doi: 10.1080/15384101.2018.1563395

    Figure Lengend Snippet: New tetracycline- and AID-controlled vectors. (a) Cloning sites of pUHD-AID. Part of the AID tag is shown (the AID tag is 228 amino acid long (~25 kDa)). The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown (except the two BamHI sites). *: termination codon. A puromycin-resistant gene is present in the construct for antibiotic selection in mammalian cells. The complete sequence of pUHD-AID can be found in the Supplemental information. (b) Cloning sites of pRevTRE-AID. The AID tag is followed by a 5xGA linker and multiple cloning sites. Only unique restriction enzyme sites are shown. *: termination codon. A hygromycin-resistant gene is expressed from this construct to allow antibiotic selection in mammalian cells. The complete sequence of pRevTRE-AID can be found in the Supplemental information. (c) Workflow for conditional gene inactivation using pUHD-AID or pRevTRE-AID. (1) Silence mutations (changing 3–4 bases) can be introduced into the cDNA to render the cDNA resistant to the CRISPR-Cas9. Alternatively, CRISPR-Cas9 that targets the endogenous gene but not the cDNA (e.g. against a intron-exon boundary) can be used. (2) Cloning sites are shown in panels A and B. (3–4) After cells are transfected with DNA or infected with retroviruses, they are selected with puromycin (pUHD-AID) or hygromycin B (pRevTRE-AID). (5) The cells are then transfected with CRISPR-Cas9 against the gene of interest. Various selection or sorting methods can be used to enrich the transfected cells. (6) Although the entire workflow can be initiated using cells stably expressing TIR1, we found that putting TIR1 into the cells at this stage generated more clones that could effectively respond to IAA. (7) Single clones are isolated and analysed for clones lacking the expression of the endogenous gene of interest, but expressing the AID-tagged version (ideally at a level similar to the endogenous protein) which can be eliminated in the presence of Dox and IAA.

    Article Snippet: Cells were treated with the following reagents at the indicated final concentration: blasticidin (5 µg/ml) (Life Technologies, Carlsbad, CA, USA), doxycycline hydrochloride (Dox) (2 µg/ml), hygromycin B (200 µg/ml) (Life Technologies), indole-3-acetic acid (IAA) (50 µg/ml) (Sigma-Aldrich), nocodazole (0.33 µM) (Enzo Life Sciences, Farmingdale, NY, USA), and puromycin (0.6 µg/ml) (Sigma-Aldrich).

    Techniques: Clone Assay, Construct, Selection, Sequencing, CRISPR, Transfection, Infection, Stable Transfection, Expressing, Generated, Isolation

    Synergy is suppressed in mutants resistant to sulphate-transport inhibition. Growth of wild type  S. cerevisiae  BY4741 or isogenic  cys3Δ/met15Δ  or  sul1Δ/sul2Δ  mutants was monitored continuously in YEPD or YNB (for Hygro+DIDS) broths. The appropriate doses of indicated agents used to test synergy were: 50 μg ml −1  paromomycin for wild type and  cys3Δ/met15Δ , 300 μg ml −1  paromomycin for  sul1Δ/sul2Δ , 150 μg ml −1  hygromycin B, 50 μM chromate, 1 mM molybdate, 1 mM DIDS. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

    Journal: Scientific Reports

    Article Title: Novel, Synergistic Antifungal Combinations that Target Translation Fidelity

    doi: 10.1038/srep16700

    Figure Lengend Snippet: Synergy is suppressed in mutants resistant to sulphate-transport inhibition. Growth of wild type S. cerevisiae BY4741 or isogenic cys3Δ/met15Δ or sul1Δ/sul2Δ mutants was monitored continuously in YEPD or YNB (for Hygro+DIDS) broths. The appropriate doses of indicated agents used to test synergy were: 50 μg ml −1 paromomycin for wild type and cys3Δ/met15Δ , 300 μg ml −1 paromomycin for sul1Δ/sul2Δ , 150 μg ml −1 hygromycin B, 50 μM chromate, 1 mM molybdate, 1 mM DIDS. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

    Article Snippet: Chemicals With the exception of hygromycin B (Panreac Applichem), all chemicals were from Sigma-Aldrich: paromomycin, streptomycin, Na2 CrO4 , Na2 MoO4 , Na3 VO4 , 4,4′-diisothio-cyanatostilbene-2,2′-disulphonate (DIDS), sodium malonate, sodium oxalate, NaHCO3 , Na2 SeO4 , Na2 S2 O3 , Na2 WO4 , Na2 S4 O6 .

    Techniques: Inhibition

    Aminoglycosides combined with sulphate transport inhibitors produce synergistic inhibition of yeast growth. Growth of S. cerevisiae BY4743 was monitored continuously in YEPD broth (or YNB broth for Hygro+DIDS, as DIDS formed precipitates in YEPD), supplemented with different aminoglycosides in combination with sulphate transport inhibitors (the most effective synergistic combinations are presented; other combinations are in Supplementary Fig. S1 ). Aminoglycoside doses were as for Fig. 1 except in combination with DIDS where hygromycin B was at 150 μg ml −1 . The sulphate transport inhibitors were chromate (Cr), 50 μM; molybdate (Mo), 1 mM, orthovanadate (Orthovan), 1 mM; DIDS, 1 mM; malonate (Mal), 50 mM (the highest sub-inhibitory dose tested); oxalate (Ox), 5 mM (the highest soluble dose attainable); bicarbonate (Bic), 7.5 mM. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

    Journal: Scientific Reports

    Article Title: Novel, Synergistic Antifungal Combinations that Target Translation Fidelity

    doi: 10.1038/srep16700

    Figure Lengend Snippet: Aminoglycosides combined with sulphate transport inhibitors produce synergistic inhibition of yeast growth. Growth of S. cerevisiae BY4743 was monitored continuously in YEPD broth (or YNB broth for Hygro+DIDS, as DIDS formed precipitates in YEPD), supplemented with different aminoglycosides in combination with sulphate transport inhibitors (the most effective synergistic combinations are presented; other combinations are in Supplementary Fig. S1 ). Aminoglycoside doses were as for Fig. 1 except in combination with DIDS where hygromycin B was at 150 μg ml −1 . The sulphate transport inhibitors were chromate (Cr), 50 μM; molybdate (Mo), 1 mM, orthovanadate (Orthovan), 1 mM; DIDS, 1 mM; malonate (Mal), 50 mM (the highest sub-inhibitory dose tested); oxalate (Ox), 5 mM (the highest soluble dose attainable); bicarbonate (Bic), 7.5 mM. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

    Article Snippet: Chemicals With the exception of hygromycin B (Panreac Applichem), all chemicals were from Sigma-Aldrich: paromomycin, streptomycin, Na2 CrO4 , Na2 MoO4 , Na3 VO4 , 4,4′-diisothio-cyanatostilbene-2,2′-disulphonate (DIDS), sodium malonate, sodium oxalate, NaHCO3 , Na2 SeO4 , Na2 S2 O3 , Na2 WO4 , Na2 S4 O6 .

    Techniques: Inhibition

    Synergistic growth inhibition of fungi pathogenic to humans. ( A ) Growth of C. albicans in YEPD broth supplemented with 200 μg ml −1 paromomycin, 10 μg ml −1 hygromycin B and/or 25 μM chromate. ( B ) Growth of C. glabrata in YEPD broth supplemented with 400 μg ml −1 paromomycin and/or 50 μM chromate. ( C ) Growth of Cryptococcus neoformans in YEPD broth supplemented with 12.5 μg ml −1 paromomycin, 0.625 μg ml −1 hygromycin B and/or 12.5 μM chromate. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

    Journal: Scientific Reports

    Article Title: Novel, Synergistic Antifungal Combinations that Target Translation Fidelity

    doi: 10.1038/srep16700

    Figure Lengend Snippet: Synergistic growth inhibition of fungi pathogenic to humans. ( A ) Growth of C. albicans in YEPD broth supplemented with 200 μg ml −1 paromomycin, 10 μg ml −1 hygromycin B and/or 25 μM chromate. ( B ) Growth of C. glabrata in YEPD broth supplemented with 400 μg ml −1 paromomycin and/or 50 μM chromate. ( C ) Growth of Cryptococcus neoformans in YEPD broth supplemented with 12.5 μg ml −1 paromomycin, 0.625 μg ml −1 hygromycin B and/or 12.5 μM chromate. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

    Article Snippet: Chemicals With the exception of hygromycin B (Panreac Applichem), all chemicals were from Sigma-Aldrich: paromomycin, streptomycin, Na2 CrO4 , Na2 MoO4 , Na3 VO4 , 4,4′-diisothio-cyanatostilbene-2,2′-disulphonate (DIDS), sodium malonate, sodium oxalate, NaHCO3 , Na2 SeO4 , Na2 S2 O3 , Na2 WO4 , Na2 S4 O6 .

    Techniques: Inhibition

    The translation error-rate is synergistically increased by combining paromomycin with sulphate mimetics. ( A ) S. cerevisiae L1494 ( ade1-14 ) cells in 10-fold serial dilutions were spotted and incubated on YEPD agar supplemented with 25 μg ml −1 paromomycin, 50 μM chromate and/or 250 μM molybdate, or to YNB agar with 100 μg ml −1 hygromycin B and/or 1 mM DIDS. ( B ) Wild type yeast transformed with the dual-luciferase plasmid 36 were exposed to 50 μg ml −1 aminoglycoside and/or 100 μM chromate, 500 μM molybdate, or 1 mM DIDS in YNB broth before determination of firefly and Renilla luciferase activities in protein extracts. The ratio of these activities indicates the level of translation read-through of the UAA stop codon separating the two ORFs. All values are means ±SEM from at least three independent determinations. RLU, relative light units. ( C ) The isogenic cys3Δ/met15Δ and sul1Δ/sul2Δ yeast double-deletants were treated as in ( B ). *p

    Journal: Scientific Reports

    Article Title: Novel, Synergistic Antifungal Combinations that Target Translation Fidelity

    doi: 10.1038/srep16700

    Figure Lengend Snippet: The translation error-rate is synergistically increased by combining paromomycin with sulphate mimetics. ( A ) S. cerevisiae L1494 ( ade1-14 ) cells in 10-fold serial dilutions were spotted and incubated on YEPD agar supplemented with 25 μg ml −1 paromomycin, 50 μM chromate and/or 250 μM molybdate, or to YNB agar with 100 μg ml −1 hygromycin B and/or 1 mM DIDS. ( B ) Wild type yeast transformed with the dual-luciferase plasmid 36 were exposed to 50 μg ml −1 aminoglycoside and/or 100 μM chromate, 500 μM molybdate, or 1 mM DIDS in YNB broth before determination of firefly and Renilla luciferase activities in protein extracts. The ratio of these activities indicates the level of translation read-through of the UAA stop codon separating the two ORFs. All values are means ±SEM from at least three independent determinations. RLU, relative light units. ( C ) The isogenic cys3Δ/met15Δ and sul1Δ/sul2Δ yeast double-deletants were treated as in ( B ). *p

    Article Snippet: Chemicals With the exception of hygromycin B (Panreac Applichem), all chemicals were from Sigma-Aldrich: paromomycin, streptomycin, Na2 CrO4 , Na2 MoO4 , Na3 VO4 , 4,4′-diisothio-cyanatostilbene-2,2′-disulphonate (DIDS), sodium malonate, sodium oxalate, NaHCO3 , Na2 SeO4 , Na2 S2 O3 , Na2 WO4 , Na2 S4 O6 .

    Techniques: Incubation, Transformation Assay, Luciferase, Plasmid Preparation

    Synergistic growth inhibition of other problem fung i. ( A ) Growth of Z. bailii after spotting 10-fold serial dilutions of cell suspension on YEPD agar supplemented with 10 μg ml −1 paromomycin, 50 μM chromate and/or 1 mM molybdate, or to YNB agar with 10 μg ml −1 hygromycin B and/or 250 μM DIDS. ( B ) Growth of R. solani on PDA agar supplemented with 300 μg ml −1 paromomycin and 15 mM molybdate. ( C ) Growth of Z. tritici in PDB medium supplemented with 0.5 μg ml −1 paromomycin, 0.25 μg ml −1 hygromycin B and/or 10 μM chromate. The data for each condition are representative of at least two independent experiments performed on different days.

    Journal: Scientific Reports

    Article Title: Novel, Synergistic Antifungal Combinations that Target Translation Fidelity

    doi: 10.1038/srep16700

    Figure Lengend Snippet: Synergistic growth inhibition of other problem fung i. ( A ) Growth of Z. bailii after spotting 10-fold serial dilutions of cell suspension on YEPD agar supplemented with 10 μg ml −1 paromomycin, 50 μM chromate and/or 1 mM molybdate, or to YNB agar with 10 μg ml −1 hygromycin B and/or 250 μM DIDS. ( B ) Growth of R. solani on PDA agar supplemented with 300 μg ml −1 paromomycin and 15 mM molybdate. ( C ) Growth of Z. tritici in PDB medium supplemented with 0.5 μg ml −1 paromomycin, 0.25 μg ml −1 hygromycin B and/or 10 μM chromate. The data for each condition are representative of at least two independent experiments performed on different days.

    Article Snippet: Chemicals With the exception of hygromycin B (Panreac Applichem), all chemicals were from Sigma-Aldrich: paromomycin, streptomycin, Na2 CrO4 , Na2 MoO4 , Na3 VO4 , 4,4′-diisothio-cyanatostilbene-2,2′-disulphonate (DIDS), sodium malonate, sodium oxalate, NaHCO3 , Na2 SeO4 , Na2 S2 O3 , Na2 WO4 , Na2 S4 O6 .

    Techniques: Inhibition

    Aminoglycosides combined with chromate produce synergistic inhibition of yeast growth. ( A ) Growth of S. cerevisiae BY4743 was monitored continuously in YEPD broth supplemented as indicated. The agents and doses were: paromomycin (Parom), 200–250 μg ml −1 ; hygromycin B (Hygro), 10 μg ml −1 ; streptomycin (Strep), 30 mg ml −1 (the highest soluble dose attainable); chromate (Cr), 50 μM. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days. ( B ) Checkerboard assays with S. cerevisiae at the indicated concentrations, according to EUCAST procedure. The growth values within the boxes are percentages of control growth (OD) determined in the absence of aminoglycoside or Cr. Shaded boxes indicate ≥50% of control growth.

    Journal: Scientific Reports

    Article Title: Novel, Synergistic Antifungal Combinations that Target Translation Fidelity

    doi: 10.1038/srep16700

    Figure Lengend Snippet: Aminoglycosides combined with chromate produce synergistic inhibition of yeast growth. ( A ) Growth of S. cerevisiae BY4743 was monitored continuously in YEPD broth supplemented as indicated. The agents and doses were: paromomycin (Parom), 200–250 μg ml −1 ; hygromycin B (Hygro), 10 μg ml −1 ; streptomycin (Strep), 30 mg ml −1 (the highest soluble dose attainable); chromate (Cr), 50 μM. Data shown are replicates from two independent cultures ± SEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days. ( B ) Checkerboard assays with S. cerevisiae at the indicated concentrations, according to EUCAST procedure. The growth values within the boxes are percentages of control growth (OD) determined in the absence of aminoglycoside or Cr. Shaded boxes indicate ≥50% of control growth.

    Article Snippet: Chemicals With the exception of hygromycin B (Panreac Applichem), all chemicals were from Sigma-Aldrich: paromomycin, streptomycin, Na2 CrO4 , Na2 MoO4 , Na3 VO4 , 4,4′-diisothio-cyanatostilbene-2,2′-disulphonate (DIDS), sodium malonate, sodium oxalate, NaHCO3 , Na2 SeO4 , Na2 S2 O3 , Na2 WO4 , Na2 S4 O6 .

    Techniques: Inhibition